Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208
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ABSTRACT |
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The protein activator of
RNA-activated protein kinase (PKR) is a proapoptotic protein called
PACT. PKR is an interferon (IFN)-induced serine-threonine protein
kinase that plays a central role in IFN's antiviral and
antiproliferative activities. PKR activation in cells leads to
phosphorylation of the -subunit of the eukaryotic protein synthesis
initiation factor (eIF)2
, inhibition of protein synthesis, and
apoptosis. In the absence of viral infections, PKR is activated
by its activator PACT, especially in response to diverse stress
signals. Overexpression of PACT in cells causes enhanced sensitivity to
stress-induced apoptosis. We examined PACT expression in
different mouse tissues and evaluated its possible role in regulating
apoptosis. PACT is expressed at high levels in colonic
epithelial cells, especially as they exit the cell cycle and enter an
apoptotic program. PACT expression also coincides with the presence
of active PKR and phosphorylated eIF2
. These results suggest a
possible role of PACT-mediated PKR activation in the regulation of
epithelial cell apoptosis in mouse colon. In addition,
transient overexpression of PACT in a nontransformed intestinal
epithelial cell line leads to induction of apoptosis, further
supporting PACT's role in inducing apoptosis.
interferon; apoptosis; protein kinase; RNA-activated
protein kinase; eukaryotic protein synthesis initiation factor 2
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INTRODUCTION |
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INTERFERONS (IFNs)
are known to have antiviral, antiproliferative, and immunomodulatory
activities, which they exert by inducing several responsive genes at
the transcriptional level (32). The double-straded
RNA-activated protein kinase (PKR), a serine/threonine kinase, is one
of the genes induced by IFNs and has been shown to be responsible for
the antiproliferative and antiviral actions of IFN (20,
37). Although its expression is induced by treatment with IFN, PKR is present at low constitutive levels in cells. The
best-studied physiological substrate of PKR activity is the -subunit
of the eukaryotic protein synthesis initiation factor eIF2, and
phosphorylation of eIF2
on Ser51 by PKR leads to
inhibition of protein synthesis (31). PKR's kinase
activity is exhibited only after its binding to an activator, and the
most well-characterized activator of PKR is double-stranded (ds) RNA
(22). Binding of an activator to PKR causes a
conformational change in PKR protein, leading to an unmasking of its
ATP-binding site and its autophosphorylation and activation
(8). On viral infection of IFN-treated cells, PKR is
activated by viral dsRNA, and this leads to a block in protein
synthesis (11, 12). PKR thus plays a central role in
antiviral activity of IFN. In addition, PKR is also involved in the
regulation of apoptosis (10, 36), cell
proliferation (14, 21), and signal transduction
(38). Overexpression or activation of PKR in HeLa
(13), COS-1 (33), U937 (39), and
NIH/3T3 (33) cells has been shown to lead to apoptosis. Mouse embryo fibroblasts from PKR knockout mice are resistant to the apoptotic cell death in response to stress signals (3). Overexpression of PKR in a tetracycline-inducible
manner and subsequent activation by dsRNA resulted in apoptosis
due to expression of members of tumor necrosis factor receptor family Fas and proapoptotic Bax (1, 4).
In all of these PKR-mediated apoptotic pathways that operate in the
absence of viral infections, the identity of a cellular activator of
PKR remained elusive until recently. We have cloned PKR activating
protein (PACT), which heterodimerizes with PKR and activates it in the
absence of dsRNA (24). PACT interacts with PKR through its
conserved dimerization domains, which are present in two copies in PKR
(25) and in three copies in PACT (24, 27).
Although the same domains are also involved in dsRNA binding,
protein-protein interactions are independent of dsRNA binding as
illustrated by the full dimerization activity of several dsRNA-binding
defective point mutants of PKR (25, 26). Like PKR, PACT is
expressed in most cell types at a very low abundance, and
overexpression of PACT causes PKR activation, leading to eIF2 phosphorylation (24). In addition, PACT-overexpressing
cells exhibit enhanced sensitivity to undergo apoptosis in
response to serum starvation and treatments with arsenite or peroxide, indicating that these stress signals elicit signal transduction pathway(s), leading to PACT-dependent PKR activation, which results in
the induction of the apoptotic cascade (9, 23).
Treatment of cells with stress agents rapidly leads to phosphorylation
of endogenous PACT, its association with PKR, and activation of PKR. Thus PACT has emerged as a stress-dependent activator of PKR
(23).
Although we have previously shown PACT's involvement in stress-induced apoptotic pathways, its role in normal tissue homeostasis has not been examined so far. In the present study, we examined the expression pattern of the mouse homolog of PACT. Our results indicate that PACT is expressed at high levels in colonic epithelial (CE) cells and that its expression pattern suggests its role in inducing CE cell apoptosis. Furthermore, a forced overexpression of PACT in normal intestinal epithelial cells leads to induction of apoptosis.
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MATERIALS AND METHODS |
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Subcloning and in vitro translation of mouse homolog of PACT. The cDNA insert from the mouse expressed sequence tag (EST) clone (GenBank accession no. AA153858) was sequenced by using T7 and T3 primers initially and then by using synthetic primers from the sequenced regions. Once sequenced, the coding region was PCR-amplified and subcloned into the XbaI and BamHI sites of BSIIKS+ vector. The [35S]methionine (Perkin-Elmer)-labeled mouse PACT protein was produced by in vitro translation using the TNT in vitro translation kit (Promega).
Immunoprecipitation analysis.
In vitro translated, 35S-labeled human and mouse PACT
proteins were synthesized by using the TNT T7-coupled reticulocyte
system from Promega. Five microliters of the in vitro-translated
35S-labeled proteins were incubated with 2 µl of
anti-human PACT polyclonal antibody in 200 µl of IP buffer (in mM):
20 Tris · HCl, pH 7.5, 100 KCl, 1 EDTA, 1 dithiothreitol, 0.2 phenylmethanesulfonyl fluoride, and 100 U/ml aprotinin, 20% glycerol,
and 1% Triton X-100 at 4°C for 30 min on a rotating wheel. Protein
A-agarose (20 µl; Boehringer-Mannheim) beads were added to the
mixture, and the incubation continued for an additional 1 h. The
beads were washed in 500 µl of IP buffer four times, the washed beads were boiled in 2× Laemmli buffer (150 mM Tris · HCl, pH 6.8, 5% SDS, 5% -mercaptoethanol, 20% glycerol) for 2 min, and eluted proteins were analyzed by SDS-PAGE on a 12% gel followed by
phosphorimager (Storm imager, Molecular Dynamics) analysis.
Western blot analysis. Total protein extracts (100 µg) from various organs of C57BL/6 mouse were analyzed by 12% SDS-PAGE and Western blot analysis as described before (23).
Immunohistochemistry.
Typically, the paraffin blocks were prepared from colon of two C57BL/6
mice and each experiment was verified by using one more set of mice.
That gave us data from four different mice for each antibody used.
C57BL/6 mice colons were removed, flushed with cold (4°C) PBS, and
opened longitudinally. Tissue samples were harvested under a dissecting
microscope and immediately fixed in 10% neutral buffered formalin,
paraffin embedded, and sectioned (5 µm) for immunohistochemistry. An
automated Techmate 500 (Biotek) stainer was used for
immunohistochemistry using the manufacturer's suggested protocol.
3,3'-Diaminobenzidine (DAB) was used as substrate for avidin biotin
complex peroxidases. Antibodies used were rabbit polyclonal anti-mouse
PKR (M550) antibody (1:500; Santa Cruz Biotechnology), rabbit
polyclonal PACT antibody (1:1,000), rabbit polyclonal phosphospecific anti-murine PKR antibody (1:500; Biosource International), rabbit polyclonal phosphospecific anti-mouse eIF2 antibody (1:500;
BioSource International), and rabbit polyclonal anti-poliferating cell
nuclear antigen (PCNA) antibody (1:1,000; Santa Cruz Biotechnology).
Negative control sections were incubated either with no primary
antibody or with preimmune serum for PACT antibody controls. Slides
were counterstained with hematoxylin, dehydrated, and mounted.
5-Bromodeoxyuridine labeling assays. To determine mitotic turnover, two C57BL/6 mice were injected intraperitoneally with 5-bromodeoxyuridine (BrdU; 30 µg/g body wt) 1 h before death. Colonic tissues were fixed in 70% ethanol and embedded in paraffin, and 5-µm sections were prepared followed by immunohistochemistry using a Signet kit (USA-HRP detection system, murine monoclonal) with anti-BrdU (1:20) antibody. The substrate used was DAB. Slides were counterstained with hematoxylin as described in the previous section.
Identification of apoptosis. Apoptotic cells were visualized in colon crypts using terminal deoxynucleotide transferase (TdT) labeling (TUNEL) assay, which detects apoptotic DNA strand breaks (6). In brief, colon segments were fixed in 4% paraformaldehyde and 5-µm paraffin sections were prepared by standard procedures. Sections were deparaffinized in xylene and incubated for 15 min with 20 µg/ml proteinase K (Qiagen), washed with PBS, and permeabilized (0.1% Triton X-100 in 0.1% sodium citrate and 0.01 M glycine). Endogenous peroxidase activity was blocked by incubating the sections in 0.3% H2O2 in methanol for 30 min, followed by washing in PBS. The TdT reaction mixture was prepared using an in situ cell death detection kit and fluorescein-labeled dNTPs (Roche) and was incubated in the dark at 37°C for 30 min before the reaction was terminated by washing in PBS. Each experiment was performed with a negative control (labeling solution without TdT). Fluorescein-labeled dNTPs were detected by using TUNEL POD (Boehringer-Manheim) incubated in the dark at 37°C for 30 min. Sections were washed in PBS and developed with 0.05% DAB and 0.03% H2O2 in PBS for 5 min, followed by counterstaining with hematoxylin.
Apoptosis assays. The rat intestinal epithelial cell line (IEC)-6 was obtained from American Type Culture Collection and cultured in DMEM with 10% fetal bovine serum, 0.1 U/ml of bovine insulin (Sigma), and penicillin/streptomycin. The Flag-PACT/pCB6+ plasmid was as described before (23, 24). Cells were cotransfected with the indicated plasmids by using the Lipofectamine (Invitrogen) reagent. Forty-eight hours after transfection, the cells were fixed in 1:1 methanol/acetone and mounted in Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI). At least 300 green fluorescent protein (GFP)-positive cells were counted as alive or dead on the basis of chromatin condensation.
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RESULTS |
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To study the expression pattern of the mouse homolog of PACT
(24), we obtained the corresponding EST clone (GenBank
accession no. AA153858) and sequenced the 1.5-kb cDNA insert. The
sequence alignment of the deduced human and murine PACT proteins is
shown in Fig. 1A. As noted,
murine PACT is highly homologous to PACT, differing only at eight
positions. Both proteins are 313 amino acids long and identical at 305 positions. Of the eight residues that are different between the two
proteins, five are conservative changes. Thus the human and mouse PACT
proteins are highly homologous. Ito et al. (9) have
reported cloning of RAX, which differs from mouse PACT only at two
positions that could be attributed to allelic variation.
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The region coding for murine PACT protein was PCR amplified from the cDNA insert and subcloned into pBSIIKS+ vector (Stratagene). We produced the murine PACT protein by in vitro translation by using this construct. As shown in Fig. 1B, murine PACT is a 34-kDa protein and is indistinguishable by mobility from human PACT. To test further whether the polyclonal antibodies raised against the human PACT will cross-react with murine PACT, we performed immunoprecipitation analysis. As shown in Fig. 1B, the antibodies cross-react with the murine PACT protein and can immunoprecipitate it efficiently. To confirm this, we also performed Western blot analysis on several human cell lines and one mouse cell line. As seen in Fig. 1C, Western blot analysis detects a 34-kDa band corresponding to PACT protein in six different human cell lines and also in the murine NIH/3T3 cells. These results show that murine PACT is highly homologous to human PACT and can be detected efficiently by using the polyclonal antibodies raised against human PACT.
To study PACT expression in different mouse tissues, protein extracts
were made from several different tissues, and Western blot analysis was
performed. As seen in Fig. 2, Western
blot analysis showed that PACT is expressed at a low level in most
tissues examined and that its expression level was the highest in colon
tissue. In skeletal muscle, the band corresponding to PACT was
undetectable, but a faint band of slightly lower molecular weight was
noted. At present, we do not know the significance, if any, of this
smaller protein. To ascertain that similar quantities of protein were analyzed in all lanes, the same blot was stripped and reprobed with
anti--actin antibody (Fig. 2, bottom). Only the spleen
lane shows a somewhat higher amount of protein loading, as judged by the intensity of
-actin signal. However, because the signal for PACT
is relatively weak in this lane, we concluded that PACT expression is
low in the spleen. Skeletal muscle and heart lanes do not show any
-actin band, because these tissues express a different actin isoform.
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Because the abundance of PACT was significantly higher in colon
compared with other tissues, we then wanted to determine the cell type
in colonic tissue that expressed PACT at higher levels. We therefore
performed immunohistochemistry on colon sections to localize PACT
expression. As seen in Fig. 3,
A and B, PACT is expressed in all cell types at a
very low abundance and at elevated levels in CE cells (brown staining,
arrows). In particular, PACT expression was highest at the top of the
crypts (arrows) and tapered off toward the base of the crypt
(arrowheads). The preimmune serum control did not show any staining at
the same dilution (data not shown). Because PACT is involved in
stress-induced PKR activation leading to apoptosis, we sought
to examine whether PKR is also expressed in CE cells. As shown in Fig.
3, C and D, PKR is expressed in the CE cells.
Compared with PACT expression, which was the highest at the luminal
surface of epithelium and the lowest at the bases of the crypts, PKR
expression pattern was relatively uniform. However, a slight increase
in expression was noted at the luminal surface. CE cells are known to
enter an apoptotic program as they migrate to the top of the
colonic crypts (5, 34). We reasoned that because PACT
levels are the highest toward the luminal surface of the colon, PKR
activation might take place selectively at this surface. This would be
consistent with the idea that PACT-induced PKR activation may lead to
apoptosis of CE cells. To determine the presence of active,
phosphorylated PKR, we stained the colon sections with antibodies
specific for the phosphorylated PKR and eIF2, which is the
physiological substrate of PKR (7, 31). We used these
antibodies for examining phosphorylation of eIF2
and PKR in response
to stress and found them to be specific for phosphorylated proteins
(23). Our immunohistochemistry analysis showed that,
although PKR levels were fairly uniform throughout the colonic crypts,
active or phosphorylated PKR was present only at the luminal (brown
staining, arrows) surface and at the top third of the crypts of colonic
crypts (Fig. 3, E and F). Consistent with this,
phosphorylated eIF2
was also found to be present primarily at the
luminal surface (Fig. 3, G and H; arrows) and the
top one-third of the crypts. eIF2
phosphorylation has also been
known to occur when cells exit the cell cycle (30). These
results strongly suggest that, as the cells migrate toward the top of
the crypts, they express higher levels of PACT, and this may lead
either by itself or in combination with other signal(s) to activation
of PKR and subsequent apoptosis. Because the proteins we are
studying here express predominantly in the luminal layer of colonic
epithelium, we wanted to ascertain that the immunohistochemical
staining observed was not due to a nonspecific "edge-effect"
staining. To confirm that the staining was specific, we did two
controls. Immunohistochemical staining performed without the primary
antibody, which showed no staining (Fig. 3I; arrowheads)
including the luminal edge. In addition, we performed the staining to
detect expression of PCNA, which is known to localize to the
proliferating cell nuclei. PCNA antibody showed no staining at the
luminal epithelial layer (Fig. 3J; arrowheads), thereby
confirming that the staining obtained in Fig. 3,
A-H is specific. As expected, the PCNA
antibody stained the nuclei of epithelial cells in the lower one-third
of the crypts, which have been shown to be actively proliferating
(5). In addition to the colon, we have also examined PACT
expression in the small intestine of mice. Although PACT is expressed
at similar levels in small intestine and is present predominantly in
epithelial cells, we do not see any gradient of expression along the
villus axis (data not shown).
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We extended these observations to study the proliferative status of CE
cells and their apoptosis. To visualize the zone of proliferation in colonic crypts, mice were injected 1 h before death with BrdU. The BrdU antibody localized to the nuclei of proliferating cells, which were located predominantly at the base of
the colonic crypts (Fig. 4B,
arrows). These results elucidate the fact that the proliferative
compartment of the colonic crypts are located mainly at the lower
one-third of the crypts and that the CE cells, located in the upper
two-thirds of the crypt region, have exited the cell cycle. These
results are in agreement with previous studies (5), which
have indicated that the proliferative compartment lies in the lower
one-third of the colonic crypt. We then performed TUNEL analysis on
colonic tissue sections to detect for CE cells carrying
fragmented DNA characteristic of apoptosing cells. As seen in Fig.
4D, these were located predominantly at the luminal surface
of the colonic crypts (brown nuclear staining, arrows). Thus the cells
at the luminal surface of the colon express a high level of PACT and
also show presence of phosphorylated eIF2 and PKR. These same cells
also stain positive for apoptosis by TUNEL assays.
Proliferating cells, on the other hand, are located mainly at the lower
one-third of the crypts. Our data, therefore, indicate that
PACT-dependent pathways may be involved, at least in part, for the
control of CE cell apoptosis. Although PACT levels seem
uniformly high at the luminal layer, the apoptotic cells, as judged
by TUNEL staining, are not as uniformly distributed. This indicates
that, in addition to PACT-dependent PKR activation, other signals may
be essential for onset of apoptotic program.
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To assay for the functional significance of the higher levels of PACT
in CE cells, we examined the effect of PACT overexpression on CE cell
survival. We cotransfected the IEC-6 cells with a flag epitope-tagged
PACT expression construct and an enhanced GFP (EGFP) expression
construct. This allows us to mark and follow the apoptosis of
the transfected population. Forty-eight hours after the transfection, the transfected cells were examined for hallmark signs of
apoptosis, such as cell shrinkage, membrane blebbing, and
nuclear condensation as seen by DAPI staining. It was observed that an
overexpression of PACT resulted in pronounced chromatin condensation
[Fig. 5, top, B, arrows]. To
quantify the percent apoptosis within the transfected
population, we counted percentage of cells showing nuclear condensation
within the EGFP-positive transfected population. Only 6% of the
vector-transfected cells underwent apoptosis [Fig. 5,
top, D, arrowheads], whereas 21% of the
PACT-overexpressing cells underwent apoptosis (Fig. 5,
bottom). This indicates a 3.5-fold increase in
apoptosis as a result of PACT overexpression. The expression of
PACT in IEC-6 cells was confirmed by Western blot analysis with the
anti-flag antibody (data not shown).
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DISCUSSION |
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Renewal of mouse intestinal epithelium takes place rapidly and continuously (34). Proliferation in the small intestine is restricted to the mucosal invaginations termed as crypts of Liberkuhn. The stem cells at the crypt bottom give rise to four epithelial lineages, three of which differentiate as they migrate from the crypt to a villus and then upward toward the lumen of the intestine (28). Once the epithelial cells reach the upper portion of the villus, they are removed by apoptosis and extrusion (17). In the colon, there are no villi and the upward migration of epithelial cells terminates with their incorporation into a hexagonal surface epithelial cuff. In normal colon, apoptosis occurs mainly at the luminal surface, after the migration and terminal differentiation of colonocytes from the base of the crypt (17). It has been suggested that any deregulation of this normal physiological apoptosis may lead to formation and growth of colonic neoplasms.
Although the transgenic and knockout mice have provided important clues about the regulation of intestinal apoptosis (35), the exact contribution of the various apoptotic pathways remains unclear. In the normal, unstressed intestine, spontaneous apoptosis occurs at a very low level at the base of the crypt at or near the position of the epithelial stem cells. Knockout mice studies indicate that this low level of apoptosis at the base of the crypts is independent of both p53 (2, 18) and Bax (29) in both small intestine and colon. Bcl2, on the other hand, has been shown to regulate spontaneous apoptosis in colon, and Bcl2 null mice show elevated levels of apoptosis at the bases of the crypts (19). Although Bax expression has been noted at the very luminal surface in colon (15), Bax null mice show normal CE cell renewal (29).
In this report, we have examined the activation of a proapoptotic
kinase, PKR, in colonic epithelium. Activation of PKR in tissue culture
cells has been shown to lead to apoptosis (1, 13, 16, 23,
33, 39). PACT is a protein activator of PKR, which is involved
in regulating PKR activation in response to stress signals (23,
24). We (23) have shown previously that treatment
of cells with stress agents leads to rapid phosphorylation of PACT
followed by its association with PKR, leading to activation of PKR's
kinase activity. PKR activation is followed by eIF2 phosphorylation
and apoptosis.
Our results, presented here, show for the first time that PACT is
expressed at high levels in CE cells. It has been shown that the upper
two-thirds of the colonic crypt represents epithelial cells that are no
longer proliferating but continue to differentiate and migrate upward
(34). By using a phosphospecific antibody for PKR and its
substrate eIF2, we have shown that the presence of activated PKR in
epithelial cells coincides with the cessation of proliferation (Fig. 3,
E-H). This correlates well with the immunohistochemical analysis of PACT expression, which appears to be
predominantly at the upper one-third of the colonic crypts (Fig. 3,
A and B) and the highest at the luminal surface.
Although PKR is present in CE cells at the bottom of the crypts,
presence of active PKR and phosphorylated eIF2
is detected only
toward the very top of the crypts, where the levels of PACT are also higher, thereby suggesting that PACT-dependent PKR activation may occur
in epithelial cells in the upper portions of the crypts. Overexpression
of PACT in normal intestinal epithelial cells led to apoptosis
(Fig. 5, A and B), thereby strengthening the
possible role of PACT in CE cell apoptosis.
Although we observe in tissue culture cells that overexpression of PACT
is sufficient to trigger an apoptotic outcome, the endogenous PACT
in normal cells is rapidly phosphorylated before PKR activation by an
as yet unidentified kinase in response to stress signals
(23). It remains to be seen whether mere overexpression of
PACT at the top one-third of the crypt may be sufficient to trigger PKR
activation. Additional signals may exist that control phosphorylation
of PACT and subsequent PKR activation, especially because every cell
that expresses high levels of PACT does not show positive TUNEL
staining. Identification of downstream pathways triggered by PKR
activation or eIF2 phosphorylation and involved in mediating CE cell
apoptosis may provide new clues to understanding the regulation
of intestinal homeostasis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Roberd Bostick and Tonia Crooks for their help in the immunohistochemical analysis, Dr. Franklin Berger and Jody Tucker for their help in TUNEL and BrDU labeling assays, and Dr. Michael Dewey for providing the C57BL/6 mice.
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FOOTNOTES |
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This work was supported, in part, by South Carolina Cancer Center Grant E183 (to R. C. Patel).
Address for reprint requests and other correspondence: R. C. Patel, Dept. of Biological Sciences, University of South Carolina, 700 Sumter St., Columbia, SC 29208 (E-mail: patelr{at}sc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 24, 2002;10.1152/ajpgi.00498.2001
Received 21 November 2001; accepted in final form 18 April 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Balachandran, S,
Kim CN,
Yeh WC,
Mak TW,
Bhalla K,
and
Barber GN.
Activation of the dsRNA-dependent protein kinase, PKR, induces apoptosis through FADD-mediated death signaling.
EMBO J
17:
6888-6902,
1998
2.
Clarke, AR,
Gledhill S,
Hooper ML,
Bird CC,
and
Wyllie AH.
p53 Dependence of early apoptotic and proliferative responses within the mouse intestinal epithelium following gamma-irradiation.
Oncogene
9:
1767-1773,
1994[ISI][Medline].
3.
Der, SD,
Yang YL,
Weissmann C,
and
Williams BR.
A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis.
Proc Natl Acad Sci USA
94:
3279-3283,
1997
4.
Donze, O,
Dostie J,
and
Sonenberg N.
Regulatable expression of the interferon-induced double-stranded RNA dependent protein kinase PKR induces apoptosis and fas receptor expression.
Virology
256:
322-329,
1999[ISI][Medline].
5.
Efstathiou, JA,
and
Pignatelli M.
Modulation of epithelial cell adhesion in gastrointestinal homeostasis.
Am J Pathol
153:
341-347,
1998
6.
Gavrieli, Y,
Sherman Y,
and
Ben-Sasson SA.
Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
J Cell Biol
119:
493-501,
1992[Abstract].
7.
Hovanessian, AG.
The double stranded RNA-activated protein kinase induced by interferon: dsRNA-PK.
J Interferon Res
9:
641-647,
1989[ISI][Medline].
8.
Hovanessian, AG,
and
Galabru J.
The double-stranded RNA-dependent protein kinase is also activated by heparin.
Eur J Biochem
167:
467-473,
1987[Abstract].
9.
Ito, T,
Yang M,
and
May WS.
RAX, a cellular activator for double-stranded RNA-dependent protein kinase during stress signaling.
J Biol Chem
274:
15427-15432,
1999
10.
Jagus, R,
Joshi B,
and
Barber GN.
PKR, apoptosis and cancer.
Int J Biochem Cell Biol
31:
123-138,
1999[ISI][Medline].
11.
Katze, MG.
The war against the interferon-induced dsRNA-activated protein kinase: can viruses win?
J Interferon Res
12:
241-248,
1992[ISI][Medline].
12.
Katze, MG.
Regulation of the interferon-induced PKR: can viruses cope?
Trends Microbiol
3:
75-78,
1995[Medline].
13.
Kibler, KV,
Shors T,
Perkins KB,
Zeman CC,
Banaszak MP,
Biesterfeldt J,
Langland JO,
and
Jacobs BL.
Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells.
J Virol
71:
1992-2003,
1997[Abstract].
14.
Koromilas, AE,
Roy S,
Barber GN,
Katze MG,
and
Sonenberg N.
Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase.
Science
257:
1685-1689,
1992[ISI][Medline].
15.
Krajewski, S,
Krajewska M,
Shabaik A,
Miyashita T,
Wang HG,
and
Reed JC.
Immunohistochemical determination of in vivo distribution of Bax, a dominant inhibitor of Bcl-2.
Am J Pathol
145:
1323-1336,
1994[Abstract].
16.
Lee, SB,
Rodriguez D,
Rodriguez JR,
and
Esteban M.
The apoptosis pathway triggered by the interferon-induced protein kinase PKR requires the third basic domain, initiates upstream of Bcl-2, and involves ICE-like proteases.
Virology
231:
81-88,
1997[ISI][Medline].
17.
Mayhew, TM,
Myklebust R,
Whybrow A,
and
Jenkins R.
Epithelial integrity, cell death and cell loss in mammalian small intestine.
Histol Histopathol
14:
257-267,
1999[ISI][Medline].
18.
Merritt, AJ,
Allen TD,
Potten CS,
and
Hickman JA.
Apoptosis in small intestinal epithelial from p53-null mice: evidence for a delayed, p53-independent G2/M-associated cell death after gamma-irradiation.
Oncogene
14:
2759-2766,
1997[ISI][Medline].
19.
Merritt, AJ,
Potten CS,
Watson AJ,
Loh DY,
Nakayama K,
and
Hickman JA.
Differential expression of bcl-2 in intestinal epithelia. Correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia.
J Cell Sci
108:
2261-2271,
1995
20.
Meurs, E,
Chong K,
Galabru J,
Thomas NS,
Kerr IM,
Williams BR,
and
Hovanessian AG.
Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon.
Cell
62:
379-390,
1990[ISI][Medline].
21.
Meurs, EF,
Galabru J,
Barber GN,
Katze MG,
and
Hovanessian AG.
Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase.
Proc Natl Acad Sci USA
90:
232-236,
1993[Abstract].
22.
Nanduri, S,
Carpick BW,
Yang Y,
Williams BR,
and
Qin J.
Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation.
EMBO J
17:
5458-5465,
1998
23.
Patel, CV,
Handy I,
Goldsmith T,
and
Patel RC.
PACT, a stress-modulated cellular activator of interferon-induced double-stranded RNA-activated protein kinase, PKR.
J Biol Chem
275:
37993-37998,
2000
24.
Patel, RC,
and
Sen GC.
PACT, a protein activator of the interferon-induced protein kinase, PKR.
EMBO J
17:
4379-4390,
1998
25.
Patel, RC,
and
Sen GC.
Requirement of PKR dimerization mediated by specific hydrophobic residues for its activation by double-stranded RNA and its antigrowth effects in yeast.
Mol Cell Biol
18:
7009-7019,
1998
26.
Patel, RC,
Stanton P,
and
Sen GC.
Specific mutations near the amino terminus of double-stranded RNA-dependent protein kinase (PKR) differentially affect its double-stranded RNA binding and dimerization properties.
J Biol Chem
271:
25657-25663,
1996
27.
Peters, GA,
Hartmann R,
Qin J,
and
Sen GC.
Modular structure of PACT: distinct domains for binding and activating PKR.
Mol Cell Biol
21:
1908-1920,
2001
28.
Potten, CS,
Booth C,
and
Pritchard DM.
The intestinal epithelial stem cell: the mucosal governor.
Int J Exp Pathol
78:
219-243,
1997[ISI][Medline].
29.
Pritchard, DM,
Potten CS,
Korsmeyer SJ,
Roberts S,
and
Hickman JA.
Damage-induced apoptosis in intestinal epithelia from bcl-2-null and bax-null mice: investigations of the mechanistic determinants of epithelial apoptosis in vivo.
Oncogene
18:
7287-7293,
1999[ISI][Medline].
30.
Proud, CG.
Protein phosphorylation in translational control.
Curr Top Cell Regul
32:
243-369,
1992[ISI][Medline].
31.
Samuel, CE.
The eIF-2 protein kinases, regulators of translation in eukaryotes from yeasts to humans.
J Biol Chem
268:
7603-7606,
1993
32.
Sen, GC,
and
Ransohoff RM.
Interferon-induced antiviral actions and their regulation.
Adv Virus Res
42:
57-102,
1993[ISI][Medline].
33.
Srivastava, SP,
Kumar KU,
and
Kaufman RJ.
Phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase.
J Biol Chem
273:
2416-2423,
1998
34.
Stappenbeck, TS,
Wong MH,
Saam JR,
Mysorekar IU,
and
Gordon JI.
Notes from some crypt watchers: regulation of renewal in the mouse intestinal epithelium.
Curr Opin Cell Biol
10:
702-709,
1998[ISI][Medline].
35.
Watson, AJ,
and
Pritchard DM.
Lessons from genetically engineered animal models. VII. Apoptosis in intestinal epithelium: lessons from transgenic and knockout mice.
Am J Physiol Gastrointest Liver Physiol
278:
G1-G5,
2000
36.
Williams, BR.
PKR; a sentinel kinase for cellular stress.
Oncogene
18:
6112-6120,
1999[ISI][Medline].
37.
Williams, BR.
Role of the double-stranded RNA-activated protein kinase (PKR) in cell regulation.
Biochem Soc Trans
25:
509-513,
1997[ISI][Medline].
38.
Williams, BRG
The role of the dsRNA-activated kinase, PKR, in signal transduction.
Semin Virol
6:
191-202,
1995[ISI].
39.
Yeung, MC,
and
Lau AS.
Tumor suppressor p53 as a component of the tumor necrosis factor-induced, protein kinase PKR-mediated apoptotic pathway in human promonocytic U937 cells.
J Biol Chem
273:
25198-25202,
1998
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