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INTRODUCTION |
The interferon (IFN)1
family of cytokines stimulate antiviral, antitumor, antiproliferative,
and immunoregulatory activities (1-3). Upon binding to receptors, IFNs
activate a signaling cascade wherein Janus tyrosine kinases
induce tyrosine phosphorylation of signal-transducing activators of
transcription proteins (4, 5). These transcription factors induce
expression of hundreds of genes that possess a wide range of activities
(6). Although a great deal is known about antiviral actions of IFNs,
mechanisms responsible for their antitumor actions are unclear.
In vivo, they up-regulate expression of tumor-specific
antigens, natural killer, and T cell function (7-9). IFNs also
activate growth suppressive proteins such as pRb (10, 11),
down-regulate c-Myc (12), and suppress activity of transcription
factor E2F (13). Protein kinase R and ribonuclease L, which inhibit
viral growth in IFN-treated cells (14, 15), also play a role in growth suppression (16, 17). The family of transcription factors known as IFN
gene regulatory factors (IRFs) also mediates the effects of IFNs (18).
Two members of this family, IRF-1 and interferon consensus
sequence-binding protein (IRF-8) are up-regulated by IFN-
. Deletion
of these genes results in myelodysplasias or chronic myelogenous
leukemia-like disease (19, 20). Because IRFs are transcription factors,
and their biological activity depends on genes they induce;
characterization of downstream gene products should allow
identification of critical regulators of growth suppression.
We have previously shown that IFN-
suppresses the growth of ovarian
tumor xenografts in nude mice (21) and that IFN-
induces apoptosis
in these cells. To identify death genes we used an antisense technical
knockout approach (22). In this approach, death regulatory genes are
identified by their ability, when expressed in antisense orientation,
to confer resistance to death inducers. Using this technique we have
identified several genes, regulators of interferon-induced death
(RIDs), that enhance IFN-
-activated death. In this study we have
characterized one of these genes, RID-2, and identified it as human
inositol hexakisphosphate kinase 2 (IP6K2) (23). IP6K2 catalyzes the
synthesis of diphosphoinositol pentakisphosphate (PP-IP5) using
inositol hexaphosphate (IP6) as a substrate in the presence of ATP.
Hence, PP-IP5 may cause growth suppression.
We show that cellular IP6K2 levels are post-transcriptionally enhanced
by IFN-
. Overexpression of IP6K2 enhances both growth suppressive
and apoptotic activities of IFN-
. A dominant negative inositol
phosphate binding domain (IPBD) mutant is highly resistant to both the
antiproliferative and apoptotic functions of IFN-
. Thus, our studies
ascribe a novel function for IP6K2 in cell growth control via apoptosis.
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EXPERIMENTAL PROCEDURES |
Reagents--
Human IFN-
(Serono), specific activity 2.7 × 108 units/mg; IFN-
2b (Schering Plough), specific
activity 3 × 108 units/mg; IFN-
(Roche Molecular
Biochemicals), specific activity 2 × 107
units/mg; [3H]inositol and [3H]IP6
(PerkinElmer Life Sciences); anti-PARP antibody (Biomol); anti-caspase
3 antibody (Pharmingen); anti-myc antibody (Oncogene Research
Products); and horseradish peroxidase goat antirabbit IgG (Pierce) were
used in these studies.
Cell Growth Assays--
Cells were treated with IFNs during
growth in RPMI 1640 and 5% fetal bovine serum. Growth was monitored
using a calorimetric assay (24). Each treatment group contained eight
replicates. Cells were fixed and stained with sulforhodamine B after 7 days. Bound dye was eluted from cells, and absorbance
(Aexp) was measured at 570 nm. One plate was
fixed 8 h after plating to determine the absorbance representing
starting cell number (Aini). Absorbance with
this plate and that obtained with untreated cells at the end of the
growth period (Afin) were taken as 0 and 100%
growth, respectively. Thus, percent control growth = 100% × (Aexp
Aini)/(Afin
Aini) expressed as a percent of untreated
controls; a decrease in cell number (death) falls on the negative
scale. To determine cell cycle distribution, cells were stained with
propidium iodide and analyzed by flow cytometry (Beckton Dickinson)
using MultiPass software.
Construction of Antisense Expression Library--
Total RNA from
NIH-OVCAR-3 cells, treated with IFN-
(500 units/ml) for 0, 1, 2, 4, 8, 16, 24, 48, and 72 h, was prepared using RNAZol B (Tel-Test).
RNAs were pooled, and total poly(A)+ RNA was isolated
(polyAttract; Promega). cDNA libraries were constructed with a
commercially available kit (Stratagene). 10 µg of mRNA was used
for preparing the cDNA library, using an oligo(dT) primer and a
dNTP mixture containing 5-methyl dCTP. After second-strand synthesis,
Pfu thermal DNA polymerase was used to create blunt-ended cDNA. cDNAs were ligated to a bifunctional linker,
5'-GCTTGGATCCAAGC-3'. Ligated to the 3' and 5' ends of the cDNA,
this linker generates HindIII and BamHI sites,
respectively (25). The library was digested with HindIII and
BamHI, purified on a Sepharose 6B column, and ligated into
an episomal vector pTKO1 (from Adi Kimchi, Weizmann Institute, Rehovot, Israel), which carried markers for
selection in eukaryotic and bacterial cells (22). When cloned into
pTKO1 the cDNA is expressed in antisense orientation. The library
was transformed into Escherichia coli DH10B, and
plasmid DNA was extracted and purified on CsCl gradients.
Electroporation of the library (40 µg) into NIH-OVCAR-3 cells
(107) was followed by selection with hygromycin B (200 µg/ml) and IFN-
(2000 units/ml) for 4 weeks. All pTKO1-transfected
cells (selected similarly) died after 14 days. After 4 weeks
selection-surviving colonies were pooled and expanded, and Hirt DNA
extracts were prepared. DNA was digested with DpnI and
electroporated into E. coli DH10B. Resultant colonies were
screened by PCR using pTKO1-specific primers to detect inserts.
Episomes (20 µg) were tested for protection against IFN-
-induced
death by electroporation into NIH-OVCAR-3 cells. Selection with IFN-
and hygromycin was initiated after 24 h.
Construction of RID-2 Mutants--
RID-2 cDNA was
digested from pTKO1 and cloned into the pCXN2myc mammalian expression
vector (26) in which the chicken actin promoter regulates expression of
the Myc-His-tagged insert. A substitution mutant (SUB) of the putative
IPBD was created using PCR-based site-directed mutagenesis (Stratagene)
with full-length RID-2 as template, replacing the highly conserved
(bold) core consensus IPBD
(PCVLDLKMG) with point mutations at 7 of 9 residues (to yield amino acid residues
ACTANLAAA) using primers
5'-pAACCTCGCGGCAGCAACACGACAACATGGTGAT-3' and
5'-pAGCGGTACAAGCCACCTCATAGCGGGAAGT-3' (p indicates phosphorylated 5'
nucleotide). PCR products were digested with DpnI, ligated,
and then transformed into JM109 E. coli, and plasmids were
isolated. An antisense mutant (ANTI) was created by ligating RID-2 open
reading frame into pCXN2myc in antisense orientation. Mutations were
confirmed by sequencing. Constructs were electroporated into
NIH-OVCAR-3 cells, and stable transfectants were selected with G418.
After 3 weeks of selection, surviving clones were pooled for further
studies. Expression of mutants was monitored by Western blotting.
Northern Blot Analysis--
Total RNA (20 µg) was separated on
1% formaldehyde-agarose gels, transferred to nylon membrane, and
probed with the 32P-labeled PCR product of RID-2 cDNA.
Western Blot Analysis--
Total cell protein (20 µg) was
separated on 10% SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride membrane. Membranes were
incubated with rabbit polyclonal antibody, made in our laboratory,
raised against full-length bacterially expressed RID-2. After washing,
membranes were incubated with anti-rabbit IgG antibody conjugated to
horseradish peroxidase and developed using ECL reagents (Amersham
Pharmacia Biotech).
Assays for Apoptosis--
Apoptotic cells were detected using a
commercially available kit (Pharmingen) and staining with annexin
V-FITC and propidium iodide (PI). Cells were analyzed by flow
cytometry. DNA fragmentation was detected using the
APO-BRDUTM kit (Pharmingen). Cells were labeled with
bromo-dUTP using terminal deoxynucleotidyltransferase, stained
with FITC-conjugated anti-bromodeoxyuridine monoclonal antibody
followed by RNase-PI. The percentage of FITC-positive cells was
determined by flow cytometry.
Inositol Hexakisphosphate Kinase Enzymatic Assay--
IP6K
activity (27) was determined in whole cell extracts of 107
NIH-OVCAR-3 cells by Dounce homogenization on ice in 100 mM
KCl, 20 mM NaCl, 1 mM EGTA, 20 mM
HEPES, pH 7.4, and adjusted to 1.5 mg of protein/ml. Using 0.15-ml
extracts, kinase assays were run in 0.5 ml of 100 mM KCl,
25 mM HEPES, pH 7.2, 5 mM Na2ATP, 6 mM MgSO4, 10 mM phosphocreatine,
20 U creatine phosphokinase, 0.1 mg of saponin using
[3H]IP6 (PerkinElmer Life Sciences) as substrate.
Reactions were incubated at 37 °C for 20 min. Reactions were
terminated on ice and then quenched with 0.25 ml of 6% v/v perchloric
acid and 0.5 mg/ml IP6 (Calbiochem) followed by extraction with 1:1
freon/octylamine and concentration by Speedvac. Products were separated
using polyethyleneimine cellulose thin-layer chromatography (PEI-TLC;
Merck). The reaction was spotted onto PEI-TLC plates and developed in
1.1 M KH2PO4, 0.8 M
K2HPO4, and 2.3 M HCl. Lanes were
divided into 1-cm fractions, and the PEI cellulose matrix was scraped
from the TLC plates, shaken with 0.5 ml of 16 M HCl, mixed
with 0.5 ml of H2O, 3 ml of scintillant, and counted.
~80% of applied 3H was recovered by this method. IP6 and
PP-IP5 migrated with an Rf migration ratio of ~0.75
and ~0.45, respectively, and comigrated with standard preparations.
[3H]PP-IP5 standard was prepared by incubation of 20 ng
of recombinant IP6K2 and [3H]IP6 in a kinase reaction as
above (60-min incubation), followed by high pressure liquid
chromatography purification on a 4.6 × 125-mm Partisphere
strong anion exchange column (Whatman). Gradient elution
utilized Buffer A (1 mM Na2EDTA) and Buffer B
(Buffer A and 1.3 M
(NH4)2HPO4). On PEI-TLC plates,
area under the curve (AUC) was used to determine total radioactivity of
each species.
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RESULTS |
IFN-
Is Cytotoxic to Ovarian Carcinoma Cells--
We
have previously shown that human IFN-
induces regression of
NIH-OVCAR-3 human ovarian carcinoma in athymic nude mice (21). To
directly demonstrate that IFN-
was cytotoxic in vitro, we treated tumor cell lines with various IFNs. After 1 week, cell growth
was measured using a colorimetric assay based on binding of the
chromophore sulforhodamine B (24). Increasing doses of IFN-
caused
significant growth inhibition in NIH-OVCAR-3 cells in vitro,
with complete cytostasis occurring at ~300 units/ml (Fig.
1A) and death occurring at
500-1000 units/ml. IFN-
was significantly more potent than IFN-
at growth inhibition. IFN-
resulted in only 39% inhibition at the
highest dose, whereas IFN-
was cytocidal. IFN-
had negligible
effect on cell growth even at 1000 units/ml (Fig. 1A). Hence
IFN-
was most effective of the type I IFNs at induction of
death.

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Fig. 1.
IFN- but not
IFN- or IFN- induces
ovarian carcinoma cell death. A, NIH-OVCAR-3 cells were
grown in the presence of 5-1000 units/ml IFN- , IFN- , or IFN- .
After 7 days cells were fixed and stained with sulforhodamine B. Absorbance (570 nm) of bound dye was measured and expressed as the
percent of untreated controls. Each data point represents mean ± S.E. of eight replicates. Values on the negative scale indicate death
of initially plated cells. B, cells were left untreated
(U) or treated with IFN- or IFN- (200 units/ml) for 2 days. Cells were stained with propidium iodide and subjected to flow
cytometry and cell cycle analysis (MultiPass).
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Microscopic examination of NIH-OVCAR-3 cells treated with IFN-
revealed a dose-dependent cytotoxicity, with condensed
nuclei detected by intense DAPI staining (Fig.
2A, arrows).
Untreated cells had a diffuse pattern of DAPI staining. To identify
apoptotic cells annexin V binding assays were performed. IFN-
was
much more efficient than IFN-
at causing phosphatidyl serine
translocation in the plasma membrane, but IFN-
did not cause
increased annexin V binding (Table
I). IFN-
at low concentrations (200 units/ml) caused 61% apoptosis after 3 days of exposure, whereas
high concentrations of IFN-
(1000 units/ml) induced only 16%
apoptosis. Maximum apoptosis induced by IFN-
(2000 units/ml) was
8%, no different from untreated cells. TUNEL assays confirmed that
chromosomal fragmentation occurred as early as 48 h after
treatment with 200 units/ml IFN-
(Fig. 2B). The degree of
chromosomal fragmentation detected by TUNEL assay mirrored the extent
of annexin V staining (Table I). IFN-
induced highest levels of
TUNEL staining, IFN-
generated intermediate levels, and IFN-
did
not cause an increase over baseline.

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Fig. 2.
IFN- induces
apoptosis in NIH-OVCAR-3 cells. A, cells were treated
with IFN- (200 units/ml) for 2 days. Condensed, DAPI-stained
apoptotic nuclei are visible (arrows). B, TUNEL
assay. Cells were treated with IFN- as above, labeled with
bromo-dUTP using terminal deoxynucleotidyltransferase, and stained with
FITC-conjugated anti-bromodeoxyuridine monoclonal antibody
(x axis) and PI (y axis). The percentage of
FITC-positive cells (in upper right and lower right
quadrants) was determined by flow cytometry.
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Table I
Effect of IFNs on induction of apoptosis in NIH-OVCAR-3 cells
Numbers indicate % of positive cells determined by flow cytometry,
mean of three separate experiments.
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To determine whether cell cycle arrest preceded death, cells were
treated with IFN-
for 1-3 days, stained with PI, and analyzed by
flow cytometry. Interestingly, neither IFN-
nor IFN-
caused cell
cycle arrest (Fig. 1B). The proportion of cells in
G0/G1, S, and G2/M phases was
unchanged after IFN-
or IFN-
treatment (200 units/ml). Thus,
death occurred independently of cell cycle arrest. Unlike IFN-
or
IFN-
, only IFN-
caused death in NIH-OVCAR-3 cells at low doses
(200 units/ml). Similarly, in WM9 melanoma and KU2 renal carcinoma
IFN-
strongly induced apoptosis compared with IFN-
(not shown).
IFN-
(2000 units/ml) did not inhibit growth or induce apoptosis in
any cell line tested (NIH-OVCAR-3, MCF-7, WM-9, KU2, MDA-MB-231, HT-29,
and A375). To confirm that caspases were activated by IFN-
, Western
blotting for caspase 3 and PARP was performed. After 8 h of
IFN-
treatment, cleavage of caspase 3 was evident, followed by PARP
cleavage at 16 h (Fig. 3,
arrows). Hence, at least one effector caspase and its
downstream substrate necessary for cell viability were proteolytically
cleaved following IFN-
treatment.

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Fig. 3.
IFN- activates
caspase 3 during apoptosis. Western blots (WB) of
caspase 3 (upper) and PARP (lower). Lysates (40 µg of protein) of NIH-OVCAR-3 cells that were treated with 0-36 h of
IFN- (200 units/ml) are shown. Caspase 3 cleavage products
(top arrow) appear earlier than PARP cleavage products
(bottom arrow).
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Genetic Approach to Isolate Cell Death Genes--
Because
NIH-OVCAR-3 cells contain non-functional p53 (28, 29), apoptosis
appears to occur via p53-independent mechanisms. We hypothesized that
hitherto unidentified molecules may transmit the death signal to the
core apoptotic machinery resulting in activation of caspases. Therefore
we sought to identify gene products using an antisense knockout
approach (22). In this technique, a death-regulatory gene can be
isolated by antisense inactivation. Cells are transfected with an
antisense cDNA library derived from tumor cells. Antisense RNA
inhibits expression of the endogenous death gene. Consequently, only
those cell clones that express the death-related antisense mRNA
will survive in the presence of death inducer. Rescued episomes are
retransfected individually into ovarian carcinoma cells to express the
antisense mRNA and eliminate false positives isolated in the first
round of transfection.
To isolate death-regulatory genes we prepared antisense cDNA
libraries cloned in the episomal vector, pTKO1. An IFN-stimulated gene
promoter drives expression of antisense RNAs in this vector. This
library was electroporated into NIH-OVCAR-3 cells (~50% transfection efficiency) and selected for resistance to hygromycin B and human IFN-
. Cell clones surviving 4 weeks of double selection were pooled,
and Hirt extracts were prepared. DNA was digested with DpnI
(to inactivate unreplicated input DNA) and electroporated into E. coli DH10B. 18 episomes were rescued in the first round. Each
episome was individually transfected into NIH-OVCAR-3 cells and
examined for cell protection against IFN-
-induced death. After two
rounds of screening, seven episomes consistently conferred resistance
to IFN-
-induced death. We named them RIDs. We chose RID-2 for
further characterization. Transfection of the RID-2 antisense episome
clearly conferred protection against IFN-
-induced death (Fig.
4). No surviving colonies were seen in
cells transfected with pTKO1 vector alone.

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Fig. 4.
Protection of NIH-OVCAR-3 cells by antisense
IP6K2 episome. Cells were electroporated with 20 µg of pTKO1
(left) or antisense IP6K2 episomes (right) and
selected for 4 weeks with IFN- and hygromycin B. Surviving cells
were fixed and stained with sulforhodamine B.
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Identification of RID-2 as IP6K2--
The RID-2 insert was
completely sequenced on both strands. The predicted open reading frame
codes for a 49.2-kDa protein. Sequence analysis (Fig.
5) revealed that this cDNA was
identical to human IP6K2, GenBankTM accession number
AAF15057 (23). A putative (Fig. 5, underlined) is located
between amino acids 201 and 232 of this protein. The IPBD motif
consensus sequence is
(LV)(LA)(DE)X(3,8)PX(VAI)(ML)DXK(ML)G, where X can be any amino acid. The most highly conserved residues are
shown in bold. This sequence is required for the catalytic activity of
inositol 1,4,5 triphosphate kinase (30). Among the notable
features are the presence of a large number of methionine residues and
the high concentration of acidic residues in the C terminus.
Henceforth, RID-2 will be referred to as IP6K2.

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Fig. 5.
Identification of RID-2 as human IP6K2.
The amino acid sequence of IP6K2, GenBankTM accession
number AAF15057, is shown. Putative IPBD is underlined with
most highly conserved residues shown in bold.
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Effect of IFN-
on Expression of IP6K2--
To study the effect
of IFN-
on IP6K2, we first examined whether its mRNA levels were
inducible. IFN-
did not induce IP6K2 mRNA in NIH-OVCAR-3 cells
even after prolonged exposure (Fig. 6A). Reprobing blots with
glyceraldehyde 3-phosphate dehydrogenase cDNA demonstrated the
presence of comparable amounts of RNA in each lane. Similarly, IP6K2
mRNA was not induced in CaOv-3 or Hey ovarian carcinoma cells (not
shown). To examine whether IFN-
enhanced expression of IP6K2
protein, Western blot analysis was performed on whole cell lysates.
IFN-
induced IP6K2 protein in a time-dependent manner.
There was no detectable induction until 4 h post-treatment.
Between 4 and 24 h, IP6K2 protein levels remained elevated (Fig.
6B). Densitometric quantitation of this blot revealed that
maximal elevation of IP6K2 protein levels occurred at 16 h of
IFN-
treatment (~5-fold induction) and declined thereafter (Fig.
6C).

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Fig. 6.
Effects of IFN- on
IP6K2 expression. (A), Northern blot (NB)
analysis. Total RNA (40 µg) derived from NIH-OVCAR-3 cells after
treatment with IFN- (200 units/ml for 0-36 h) are shown; blots were
hybridized with IP6K2 cDNA probe and glyceraldehyde-3-phosphate
dehydrogenase as control. B, Western blot
(WB) analysis of whole cell lysates (70 µg) prepared after
IFN- treatment as above. Blots were probed with anti-IP6K2 antibody
and anti-actin as control. C, densitometric quantitation of
IP6K2 protein levels from B. The x axis indicates
time of IFN- exposure (0-36 h), and the y axis indicates
band intensity normalized to actin.
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Effect of Antisense IP6K2 mRNA on Protein
Expression--
Because the rescued episome clearly conferred
protection from death (Fig. 4), it was important to confirm that this
effect was because of expression of antisense IP6K2 mRNA and
subsequent down-regulation of IP6K2 protein. Northern blot analysis
(Fig. 7) of cells transfected either with
vector alone (V) or antisense IP6K2 (ANTI) was performed. In
vector cells only one band, representing endogenous
(E) IP6K2 mRNA was seen. ANTI cells also express
endogenous mRNA and, in addition, express another mRNA species
that migrated more slowly (arrow). This second band, absent
in vector cells, corresponds to antisense IP6K2 mRNA transcribed
from the trans gene. We then determined whether anti-mRNA
expression resulted in down-regulation of protein. Western blot
analysis showed that ANTI cells contained lower levels of IP6K2 protein
compared with vector cells (Fig. 7).

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Fig. 7.
Expression of antisense IP6K2 mRNA
reduces IP6K2 protein levels. Upper panel, Northern
blot (NB) analysis of pools (~100 clones each) of
NIH-OVCAR-3 cells stably transfected with vector (V) or
antisense IP6K2 (ANTI). The arrow indicates the
position of mRNA in transgene-expressing cells. E
indicates endogenous mRNA. Lower panel, IP6K2 Western
blot (WB) of same cells and actin as control.
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IFN-
Induces IP6K Enzymatic Activity--
We next determined
whether the rise in IP6K2 protein levels following IFN-
correlated
with an increase in IP6K enzymatic activity. In vitro kinase
assays utilizing [3H]IP6 as substrate were performed to
measure IP6K activity in whole cell homogenates of untreated and
IFN-
-treated cells, using equal amounts of total cell protein. In
the reaction, PP-IP5 was synthesized by IP6K, using ATP and
[3H]IP6 as substrates. Indeed, enzymatic activity of IP6K
was increased after 4 h of IFN-
exposure, reaching a peak at
8 h and beginning to decrease from maximum by 24 h (Fig.
8). Enzymatic activity was consistent
with IP6K2 protein concentration as determined by Western blotting
(Fig. 6C). When normalized for immunoreactive IP6K2,
enzyme-specific activity was unchanged by IFN-
treatment. Untreated
cells displayed an activity of 8 ± 2 pmol/mg protein/min. IFN-
2b or IFN-
did not enhance IP6K activity at concentrations up
to 1000 units/ml (not shown). ANTI cells expressing antisense IP6K2
mRNA assayed at the 8-h time point displayed reduced enzymatic activity compared with untransfected cells, indicated by reduced (~2-fold) levels of the PP-IP5 product.

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Fig. 8.
Time course of IP6K activity in
IFN- -stimulated NIH-OVCAR-3 cells. Cells
were treated with IFN- (200 units/ml). Numbers next to
curves in top and middle panels (0, 4, 8, and 24) indicate treatment duration (h). IP6K enzymatic activity in
whole cell homogenates was determined using [3H]IP6 as
substrate in kinase reactions in vitro and detected by
PEI-TLC. Total radioactivity contained in the [3H]PP-IP5
product, represented as AUC, for IFN- -treated cells at various time
points was 70 (0 h), 120 (4 h), 209 (8 h), and 101 (24 h). Lower
panel, empty vector (V) and antisense IP6K2
(ANTI)-expressing cells. AUC for V cells was 72, and for
ANTI cells it was 32. Arrows indicate Rf for
PP-IP5 and IP6.
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A Mutant IP6K2 Inhibits IFN-
-induced Cell Death--
As
described above (Fig. 5) IP6K2 possesses a putative IPBD. Therefore, we
examined the functional relevance of this domain by generating an SUB.
In this mutant, 7 of 9 conserved residues in the IPBD were mutated at
positions 216, 218-220, and 222-224. Expression of this mutant open
reading frame in the pCXN2myc vector allowed detection of the SUB
protein with anti-Myc antibody. This vector was transfected into
NIH-OVCAR-3 cells, and stable cell lines were established. Full-length
(FL) IP6K2 was cloned into the same vector. During generation of FL
stable transfectants, 70% fewer clones were isolated, and they
exhibited growth rates of 50% compared with empty vector transfectants
(not shown). Expression of the SUB mutant was confirmed by Western blot
(Fig. 9). Comparable amounts of each
protein were present in both cell lines. No Myc-tagged proteins were
detected in cells expressing empty vector.

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Fig. 9.
Expression of mutant IP6K2 in NIH-OVCAR-3
cells. Lysates (80 µg) from cell lines were separated on
SDS-polyacrylamide gel electrophoresis and probed with antibody
directed against Myc tag (Invitrogen). Labels indicate plasmids
transfected into cells. V, pCXN2myc vector; FL,
full-length IP6K2. Predicted molecular mass (kDa) of tagged
IP6K2 proteins is 51.7 for FL and 51.5 for SUB. Western blots
(WB) were stripped and reprobed with actin as control.
Tagged proteins were detected in FL and SUB but not in vector
(V) cells.
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Growth assays were performed in NIH-OVCAR-3 cells that expressed
various constructs. In each case, pools of clones (~100 clones) were
used, so changes in growth characteristics could not be attributed to
clonal effects. As expected, the FL IP6K2-expressing cell line displayed enhanced suppression of growth in response to IFN-
(Fig.
10). The SUB mutant was resistant to
antiproliferative effects of IFN-
. SUB cells were inhibited only
45% by 100 units/ml IFN-
compared with empty vector cells, which
were inhibited 78%. At doses of IFN-
that killed vector cells
(500-1000 units/ml) SUB cells still grew, albeit slowly. As expected,
cells that expressed IP6K2 mRNA in antisense orientation (ANTI)
were most resistant to IFN-
, displaying only 34% growth inhibition
at 1000 units/ml, the highest dose tested (Fig. 10). Despite the near
total suppression of IP6K enzymatic activity in ANTI cells (Fig. 8),
their growth was still partially suppressed by IFN-
(Fig. 10),
suggesting that additional factors may mediate the antiproliferative
effects of IFN-
in these cells.

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Fig. 10.
Effect of IFN- on
growth of NIH-OVCAR-3 cells expressing SUB mutant. Growth assays
were performed as described in legend for Fig. 1. Each point
represents mean ± S.E. of eight replicates. Notations are similar
to those in Fig. 9. Cells expressing ANTI IP6K2 mRNA were most
resistant to IFN- ; cells overexpressing full-length IP6K2 were most
sensitive.
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Induction of apoptosis by IFN-
was measured using annexin V assays
(Fig. 11A). Cells
overexpressing FL IP6K2 demonstrated the highest percentage of
apoptosis following treatment with IFN-
(200 units/ml). Annexin V
staining was ~2-fold higher in FL cells (86.4%) compared with
untransfected cells (41.3%) or vector-transfected cells (40.9%).
Expression of FL IP6K2 did not induce apoptosis in the absence of
IFN-
but rather sensitized cells to death induction by IFN-
.
Importantly, SUB cells displayed decreased IFN-
-induced apoptosis
(18%) when compared with vector cells, a reduction of 55%. Therefore,
an intact IPBD is critical for induction of apoptosis.

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Fig. 11.
Apoptosis and IP6K enzymatic activity in
transfected cell lines. A, U indicates
untransfected NIH-OVCAR-3 cells. Other notations are similar to those
in Fig. 9. Cell lines were treated with IFN- (200 units/ml) and
subjected to annexin staining. The percentage of apoptotic cells was
determined by flow cytometry. B, in parallel, IP6K enzymatic
activity was determined in whole cell lysates. IP6K enzyme activity is
represented as total area under PP-IP-5 curve. Annexin V and enzymatic
data are shown as mean ± S.E. of three separate
experiments.
|
|
Finally, we examined whether ablation of IFN-
-induced apoptosis by
the IPBD mutant was because of a decrease in enzymatic activity of
IP6K. In vitro IP6 kinase assays were performed to detect
these differences (Fig. 11B). Total radioactivity
incorporated into the PP-IP5 product was expressed as AUC. Cells that
expressed FL IP6K2 had the highest enzymatic activity (235). Compared
with FL cells, SUB cells had an ~8-fold lower level of activity (30). Thus, the SUB construct functioned as a dominant negative mutant. Antisense ANTI cells had the lowest level of activity (27), which was
3-fold less than activity of vector cells (83). Thus, ablation of IP6K
enzymatic activity correlated well with loss of growth suppression and
blunted induction of apoptosis by IFN-
. These data confirm the role
of IP6K2 as a mediator of growth inhibition and apoptosis in response
to IFN-
treatment.
 |
DISCUSSION |
IFN-
was the most potent inducer of cell death of the type I
IFNs. IFN-
did not cause death at comparable doses. These
observations suggest that type I and type II IFNs employ distinct
pathways to exert anti-tumor actions. The fact that IFN-
induced
death independently of cell cycle arrest suggests that different gene products regulate growth arrest and apoptosis. Because NIH-OVCAR-3 cells lack functional p53 (28, 29), it appears that IFN-
-induced apoptosis occurs via p53-independent mechanisms. Furthermore, because
caspases are activated by a variety of death stimuli (fas, tumor
necrosis factor, perforins, and many others) it is unclear what
molecules modulate the core apoptosis machinery in response to
IFN-
.
To this end, we employed an antisense technical knockout strategy (22)
and identified the RIDs. The library used was generated using mRNA
isolated from untreated cells, as well as those treated with IFN-
.
Thus, genes expressed in all stages of apoptosis were included in the
library. Inactivation of Janus tyrosine kinases or signal-transducing
activators of transcription cannot account for the observed protection,
because expression of antisense inserts is driven by an IFN-stimulated
promoter (22). Indeed, cDNAs corresponding to these signaling
components have not been rescued in our studies (not shown). The RID-2
cDNA characterized in this study is identical to IP6K2. Because
IFN-
enhances protein levels but not mRNA levels, it appears
that a post-transcriptional regulatory mechanism controls IP6K2
expression. The protective effect of antisense IP6K2 was because of a
down-regulation of IP6K2 protein and enzymatic activity. Similarly,
expression of SUB also conferred resistance to IFN-
-induced apoptosis.
Inositol PP include PP-IP5 (7 phosphates/inositol),
bis-PP-IP4 (8 phosphates/inositol) (31), PP-IP4 (6 phosphates/inositol), and bis-PP-IP3 (7 phosphates/inositol)
(32). The specific functional roles of PP-IP5, bis-PP-IP4,
and their precursor, IP6, are not clearly understood. IP6 was
previously believed to be an inactive metabolite. Several laboratories
have demonstrated that IP6 functions as a substrate in mammalian cells,
yeast, and protozoa (23, 31, 33) to generate a family of high energy
pyrophosphates. IP6 is phosphorylated to yield PP-IP5, which in
mammalian cells is subsequently phosphorylated by a second kinase to
yield bis-PP-IP4 (34). The fact that basal IP6 concentration
in cells is relatively high and that IP6 functions as a substrate in
synthesis of small pools of high energy pyrophosphates of short
half-life (35) suggests that these pyrophosphates play an important
regulatory role. IP6 is not the only substrate for IP6K1 and IP6K2.
Both IP6Ks can also phosphorylate IP5 to yield PP-IP4 (32). Because PEI-TLC could not clearly resolve PP-IP5 and PP-IP4, it is possible the
latter compound may also contribute to growth inhibition and apoptosis.
The inositol pyrophosphates do not appear to play a role in the
metabolism of membrane-associated inositol phospholipids
(phosphoinositides), responsible for Ca2+ release and
protein kinase C activation. Links between phospholipid metabolism and
cell growth regulation have recently been identified. The type 3 inositol 1,4,5-trisphosphate receptor actively participates in
apoptosis during differentiation (36). In these studies, reduction of
type 3 inositol 1,4,5-trisphosphate receptor expression by antisense
oligonucleotides selectively blocked apoptosis. Inositol polyphosphate
4-phosphatase mediates hydrolysis of phosphatidylinositol 3,4-bisphosphate. Overexpression of this enzyme markedly reduces growth
of NIH3T3 fibroblasts (37). Our studies implicate inositol pyrophosphates as regulators of IFN-
-induced apoptosis.
IP6K1 and IP6K2 have recently been cloned (23). Although biochemically
and genetically distinct from IP6K2, IP6K1 may also play a role in
growth regulation. Expression of the IP6K2 dominant negative SUB mutant
abrogated only ~50% of growth inhibition and apoptosis induced by
IFN-
. ANTI cells and SUB cells both demonstrated an ~70%
inhibition of IP6K enzymatic activity. Yet ANTI cells were more
resistant than SUB cells to IFN-
-mediated growth inhibition, suggesting that additional, IP6K2-independent pathways may be inhibited
in the ANTI cells. It remains to be determined whether blockade of both
IP6K1 and IP6K2 function further increases the IFN-
resistance of
these cells.