From AstraZeneca Pharmaceuticals, Enabling Sciences and Technology, Wilmington, Delaware 19803
Received for publication, May 1, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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A novel human inhibitor of apoptosis protein
(IAP) family member termed Livin was identified, containing a
single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING
finger domain. The mRNA for livin was not detectable by
Northern blot in most normal adult tissues with the exception of the
placenta, but was present in developmental tissues and in several
cancer cell lines. Highest levels were observed in two melanoma-derived
cell lines, G361 and SK-Mel29. Transfection of livin in
HeLa cells resulted in protection from apoptosis induced by expression
of FADD, Bax, RIP, RIP3, and DR6. Similar to other IAP family members,
the anti-apoptotic activity of Livin was dependent on the BIR domain.
Livin was also capable of inhibiting DEVD-like caspase activity
triggered by tumor necrosis factor- The inhibitor of apoptosis protein
(IAPs)1 family is
characterized by one or more repeats of a highly conserved ~70 amino
acid domain termed the baculoviral IAP repeat (BIR) and suppress
apoptosis triggered by a wide variety of stimuli, including viral
infection, chemotherapeutic drugs, staurosporin, growth factor
withdrawal, and by components of the tumor necrosis factor- The BIR domain forms a novel zinc-fold that is the critical motif for
their anti-apoptotic activity and interaction with caspases (15). While
many IAPs contain up to three tandem BIR repeats, a single BIR domain
is sufficient for caspase interaction and protection from apoptosis
(16). Many of the IAP proteins (c-IAP1, c-IAP2, XIAP, as well as viral
and insect IAPs) also contain a RING domain near their COOH termini.
The role for the RING domain varies depending on the IAP and/or the
apoptotic stimulus, but does not appear to be required for the
anti-apoptotic activity of human IAPs (10, 16-18). Deletion of the
RING domain in c-IAP2 has suggested a critical role in TNF- Several of the IAP family members have been reported to play a role in
pathological conditions, particularly neurodegenerative disorders and
cancer. For instance, the NAIP gene was originally identified based on its deletion in patients with spinal muscular atrophy, a neurodegenerative disorder characterized by motor
neuron depletion through apoptosis (21). The correlation between NAIP, spinal muscular atrophy, and apoptosis suggests that NAIP may be
required for the survival of these neurons and that mutations in the
NAIP locus contribute to spinal muscular atrophy. In
addition, NAIP levels are transiently elevated following ischemia and
damage can be inhibited by overexpression of NAIP in vivo
(22). Expression of other IAP family members have been correlated with
cancer. For example, XIAP and c-IAP1 are found in most cancer cell
lines (23). Survivin is overexpressed in nearly all human tumors and transformed cell lines, but is rarely present in normal adult tissues
(4, 11, 24-27). Survivin is induced by angiogenic factors such as
vascular endothelial growth factor, fibroblast growth factor, and
angiopoietin-1 which may explain its elevated levels in tumors (28,
29). Depletion of Survivin using antisense or dominant negative mutants
induces apoptosis implying that Survivin expression contributes to the
survival of cancer cells (24, 30-33). Tumors expressing Survivin, as
well as other anti-apoptotic signal generally have a poorer prognosis,
likely due to their resistance to classical chemotherapy (25).
Here, we attempted to identify novel members of the IAP family via
homology searches. One gene was identified which we termed livin that encodes a protein with a single BIR domain and a
COOH-terminal RING domain. Expression of Livin inhibited apoptosis by a
number of stimuli, whereas an antisense construct was shown to induce apoptosis. Like its other family members, Livin was capable of binding
to caspases and could inhibit the proteolytic processing of caspase-9
in vitro. Restricted expression of livin mRNA
during development and transformed cell lines suggests a very specific role for Livin.
Plasmid Construction--
A partial cDNA sequence of
livin was identified in the proprietary Incyte LifeSeq data
base (accession number 1419118). To obtain the full-length construct we
screened an adult kidney cDNA library (Life Technologies, Grand
Island, NY) with a livin-specific probe,
5'-CCTTCTATGACTGGCCGCTGA-3', using the GeneTrapperTM
cDNA positive selection system (Life Technologies, Grand Island, NY). The coding sequence of livin was PCR cloned into
pcDNA3.1/V5/His-TOPO (Invitrogen, Carlsbad, CA) in both the sense
and antisense orientations. Livin was subsequently subcloned by PCR
into the BamHI/EcoRI sites in pGEX-6P (Amersham
Pharmacia Biotech, Piscataway, NJ) for purification of a glutathione
S-transferase fusion protein. Deletion mutants of Livin
( Production of Recombinant Livin--
pGEX-6P-livin was
inoculated overnight in Escherichia coli BL21 and
subsequently stimulated with 0.5 mM
isopropyl-1-thio- Maintenance of Cell Lines--
HeLa, G361, SK-Mel29, HMCB, A375,
WM115, HT144, and SW480 cell lines (ATCC, Rockville, MD) were cultured
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and penicillin (100 units/ml)/streptomycin (0.1 mg/ml).
The cells were maintained at 37 °C, 5% CO2.
Transfections were performed using LipofectAMINE Plus (Life
Technologies, Grand Island, NY) according to the manufacturer's specifications.
Purification of RNA and Northern Blotting--
Poly(A) RNA was
isolated from human melanoma cell lines (SK-Mel29, HMCB, A375, WM115,
and HT144) using the Poly(A) Pure kit (Ambion, Austin, TX) according to
the manufacturer's specifications. The RNA was denatured in sample
buffer (2.2 M formaldehyde, 50% formamide, 50 mM MOPS (pH 7.0), and 1 mM EDTA) heated at
65 °C for 10 min, electrophoresed in a 1% agarose gel containing
2.2 M formaldehyde, 50 mM MOPS (pH 7.0), and 1 mM EDTA, and then transferred by capillary elution onto
Hybond-N nylon filters (Amersham Pharmacia Biotech). In addition, we
used commercially available human RNA blots, prepared from adult and
fetal tissues and cancer cell lines (CLONTECH, Palo
Alto, CA). The blots were hybridized to random primed radiolabeled
livin (full-length cDNA containing 5'- and 3'-untranslated region) or actin
(CLONTECH, Palo Alto, CA) incubated in Hybrisol I
(Oncor, Gaithersburg, MD) overnight at 42 °C. The blots were then
washed with 2 × SSC + 0.05% SDS at room temperature followed by
high stringency washing, 0.1 × SSC + 0.1% SDS at 50 °C, and
visualized using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
GFP Viability Assays--
Viability was assessed using the green
fluorescent protein (GFP) marker produced from pTracer-SV40-derived
vectors. Transfected cells were fixed with 2% paraformaldehyde in
phosphate-buffered saline, counterstained with 10 µg/ml propidium
iodide, 200 µg/ml RNase A, and 0.1% Tween 20 for 30 min at room
temperature, and then mounted on microscope slides with Immu-mount
(Shandon, Pittsburgh, PA). A laser scanning cytometer (CompuCyte,
Cambridge, MA) was used to determine the percent of GFP-positive cells.
The propidium iodide staining served as a marker required for gating
the cell population. Relative fluorescence values were determined with an excitation at 488 nm using an argon laser and emission filters at
505-540 nm (GFP) and 614-639 nm (propidium iodide).
TUNEL Assay--
The ability of antisense against
livin to induce apoptosis was examined using
TdT-mediated dUTP-X nick end labeling (TUNEL). Transiently transfected
cells were stained with fluorescein isothiocyante-conjugated dUTP
according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN). The cells were then counterstained with 10 µg/ml propidium iodide and 200 µg/ml RNase A, and collected on microscope slides by a cytospin. The percent of TUNEL-positive cells
was evaluated using the laser scanning cytometer as described above for
GFP.
Caspase Activity Assays--
Caspase activity was measured using
the ApoAlertTM caspase-3 fluorescent assay kit
(CLONTECH). Cytosolic lysates were prepared from
transiently transfected cells and incubated with 50 µM of the fluorescent substrate 7-amino-4-trifluoromethyl coumarin (AFC) conjugated to the caspase cleavage site Asp-Glu-Val-Asp (DEVD) for
1 h at 37 °C. Hydrolyzed AFC was detected using a
CytoFluorTM 4000 fluorometer (PerSeptive Biosystems,
Framingham, MA) with peak excitation/band width at 360/40 nm and peak
emission/band width at 530/30 nm. In vitro processing of
caspase-9 was performed using [35S]methionine-labeled
caspase-9 and Apaf-1 prepared using the TNT T7 reticulocyte lysate
system (Promega Corp., Madison, WI). The proteins were desalted and
exchanged into buffer A (20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM dithiothreitol) using a
Bio-spin P-6 column (Bio-Rad, Hercules, CA). Caspase-9 (2 µl) and
Apaf-1 (6 µl) were combined with 10 µM cytochrome
c and 1 mM dATP (Sigma) and 50 µM
recombinant Livin for 1 h at 30 °C. The samples were
boiled in Laemmli buffer, and resolved by 4-20% SDS-PAGE. Gels were
fixed in 50% methanol, 10% acetic acid for 1 h, dried, and then
visualized using a PhosphorImager.
In Vitro Binding Assay--
In vitro binding
reactions were performed by combining recombinant active caspase-3 or
-7 (PharMingen, San Diego, CA) with [35S]methionine-labeled Survivin or Livin (wild-type and
deletion mutants), prepared using the TNT T7 reticulocyte lysate
system. Samples were incubated with polyclonal antibodies against
either caspase-3 or -7 (PharMingen, San Diego, CA) in 0.5 ml of NETN buffer (20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, and 0.2% Nonidet P-40) for 1.5 h at 4 °C
followed by protein G-Sepharose (Amersham Pharmacia Biotech) for 30 min. All samples were then washed three times in NETN buffer, resolved
by SDS-PAGE, and detected using a PhosphorImager.
Binding kinetics were obtained using the BiacoreTM 3000 (Biacore, Piscataway, NJ). Recombinant Livin was amine coupled
to a CM5 sensor chip and 1-500 nM active caspases-3, -7, and -9 were passed through the chip. Binding constants were determined
using the BIAevaluation software (version 3.0).
Immunoprecipitations and Western Blotting--
Associations with
caspase-9 were shown using HeLa cells transiently transfected with
either pcDNA3.1/V5/His-TOPO-livin or pcDNA3.1-myc-survivin and
treated with TNF-
Western blots were performed to confirm that the antisense construct of
livin could reduce Livin expression, but not Survivin. Transfected HeLa cells were maintained in 100 µM ZVAD-fmk
(Enzyme Systems, Livermore, CA) to prevent apoptosis. Cell lysates were prepared in Laemmli buffer and Western blots were performed as described above using antibodies against V5, Survivin (R & D Systems, Minneapolis, MN), and glyceraldehyde-3-phosphate dehydrogenase (Chemicon International, Inc., Temecula, CA).
Immunofluorescence--
The subcellular localizations of Livin
and Survivin were assessed by indirect immunofluorescence from HeLa
cells transfected with epitope-tagged expression constructs.
Twenty-four hours post-transfection the cells were fixed with 2%
paraformaldehyde and then permeabilized with 0.2% Triton X-100 in
phosphate-buffered saline. Coverslips were incubated with antibodies
against the V5 or Myc (Oncogene Research Products, Cambridge, MA)
epitopes for 1 h at 37 °C. Staining was detected using a
fluorescein isothiocyante-conjugated goat anti-mouse antibody (Life
Technologies, Grand Island, NY) for 1 h at 37 °C. Cells were
visualized by epifluorescence using an Olympus AX70 microscope equipped
with a Kodak DCS 520 digital camera (Hitech Instruments, Inc.,
Edgemont, PA).
Identification and Expression of Livin--
A homology search of a
proprietary Incyte data base using BLAST, revealed an 840-nucleotide
sequence present in a fetal kidney library and predicted to encode a
novel protein with a BIR domain and a COOH-terminal RING finger domain
(accession number 1419118). To obtain the full-length gene, we used the
GeneTrapperTM cDNA positive selection system to
screen an adult kidney cDNA library. A low abundant clone was found
which contained an in-frame stop codon in the 5'-untranslated region,
suggesting it was a full-length gene. This gene, which we termed
livin, was 1297 base pairs and predicted to encode a
280-amino acid protein (Fig.
1A; GenBank accession number
AF311388). Genomic sequence corresponding to the livin
cDNA was found in the EMBL data base (accession number AL121827)
and localized on chromosome 20. The overall protein identity of
Livin to other IAP family members based on the GAP pairwise
sequence alignment (GCGTM, Madison, WI) was 24.1%
to c-IAP1, 26.1% to c-IAP2, 34.7% to XIAP, 25.5% to NAIP, and 26.3%
to Survivin. At a structural level it was similar to Survivin with
respect to having just a single BIR domain (Fig. 1B).
However, Livin did not have a coiled-coil domain like Survivin, but
rather contained a COOH-terminal RING domain found in c-IAP1, c-IAP2,
and XIAP.
The tissue distribution of livin was studied by
Northern blotting with mRNA prepared from human adult and
developmental tissues as well as several cancer cell lines.
Using the entire cDNA sequence of livin as a
probe, three distinct mRNAs were detected with approximate sizes of
1.4, 2.0, and 2.8 kilobases (Fig. 2). The
smaller of the transcripts was consistent with the cDNA isolated in
our screen. The sizes of the three transcripts were distinct from other
IAP family members, suggesting that they were specific for
livin. Despite the presence of livin in fetal and
adult kidney cDNA libraries, within the normal tissues tested here,
livin was found only in placenta and fetal brain. It is
likely that livin is expressed transcriptionally in other
tissues, but at levels too low to be detected by the Northern blot.
Elevated levels of livin were seen in cancer cell lines,
particularly in the melanoma cell lines G361 and SK-Mel29, and to a
lesser extent, in HeLa (Fig. 2). Interestingly, the segments of the
chromosome corresponding to the livin locus displays
increased copy number in nearly all melanoma cell lines and primary
tumors (35). While livin has a narrower distribution than
survivin in cancer cells, the general patterns of
expression between these genes are similar, with no detectable
expression in normal adult tissues and elevated levels in
placenta, developing tissues, and cancer cell lines (4, 11).
Anti-apoptotic Activity of Livin--
While the BIR motif can
confer an anti-apoptotic signal via caspase interactions, some BIR
motifs are not as effective at suppressing apoptosis, and there are
BIR-containing proteins with no apparent anti-apoptotic function
(16, 18, 36). Therefore, not all BIR domain-containing proteins may be
defined properly as an IAP. The potential anti-apoptotic activity of
Livin was investigated with respect to several pro-apoptotic
signals acting at different points within the apoptotic process,
including DR6, FADD, RIP, RIP3, and Bax. DR6 is a member of the TNF
receptor family and thus is at the most upstream level of the apoptotic process (37). FADD (38), RIP (39), and RIP3 (40, 41) are adapter
proteins within the TNF-
To determine the regions of Livin necessary for its anti-apoptotic
activity, three deletion mutants comprising the
NH2-terminal ( Livin Inhibits Caspase Activity and Binds to Caspase-3, -7, and
-9--
Several IAP family members including XIAP, c-IAP1, and c-IAP2
directly bind to pro-caspase-9 and prevent its processing (14). An
association has also been observed between pro-caspase-9 and a cleaved
product of XIAP containing its BIR3 and RING domains, and therefore
presumably has a similar structure to Livin (44). Possible interactions
between Livin and/or Survivin with caspase-9 were tested in HeLa cells
transfected with these IAP family members. The cells were subsequently
treated with TNF-
The effect of Livin on caspase-9 processing was tested in
vitro. Incubation of procaspase-9 with Apaf-1, cytochrome
c, and dATP can induce the processing of caspase-9, which
can be inhibited by XIAP, c-IAP1, and c-IAP2 (14). Here, we found that
addition of recombinant Livin directly inhibited caspase-9 processing
as seen by a decrease in the 35-kDa fragment (Fig. 4C).
Thus, Livin appears to act on caspase-9 in a similar manner to XIAP,
c-IAP1, and c-IAP2 but distinct from Survivin.
The effect of Livin on DEVD-like caspase activity was tested in HeLa
cells treated with TNF-
Next, we tested whether Livin and Survivin could bind to the active
forms of caspase-3 and -7. Interactions between Survivin and caspases
have been controversial. Initial studies showed that survivin prepared
from whole cell lysates could bind to caspase-3 and -7 and also inhibit
DEVD-like caspase activity (11), and there was one report that
recombinant survivin could directly inhibit the activity of caspase-3
(12). While subsequent studies confirmed that Survivin was capable of
binding to caspase-3 directly, in whole cell preparations Survivin
bound preferentially to Cdk4 (13). Also, in a recent analysis of the
x-ray crystal structure of Survivin, the authors found no direct
interaction or inhibition of caspase-3 (46). In our experiments,
[35S]methionine-labeled Livin and Survivin were prepared
by in vitro transcription/translation and were incubated
with recombinant active caspase-3 and -7 followed by caspase specific
antibodies. Both caspase-3 and -7 interacted with Livin and Survivin,
but not with the deletion mutant
While we found that Livin and Survivin could interact with caspases-3
and -7, we have yet to find any direct effect on inhibiting the
activity of these caspase using the DEVD-AFC substrate (using up to a
1000-fold molar excess of IAP). Thus, there was an apparent discrepancy
between the relatively strong binding between Livin and caspases-3 and
-7 yet a lack of any effect on enzyme activity. In fact, other reports
of IAP inhibition of caspase activity have been reported only when
using a large (more than 5000) molar excess of IAP to caspase (10).
Perhaps, in some cases either complexes with other unidentified
proteins and/or post-translational modifications could enhance the
inhibitory effects of Livin and Survivin on caspase activity.
Subcellular Localization of Livin--
Differential subcellular
localization of the IAP family members have implied distinct roles in
apoptosis regulation (18, 32). Staining for Livin was performed using
antibodies against the V5 epitope tag in transfected HeLa cells. Livin
was observed predominantly in the nucleus and in a filamentous pattern
throughout the cytoplasm (Fig. 6).
Furthermore, transfected Survivin, which was stained using antibodies
against the Myc epitope, was observed in the same pattern as Livin
(Fig. 6). Previous studies have shown that endogenous Survivin
translocates to the nucleus during apoptosis and interacts with the
cell cycle regulator Cdk4 (13). Survivin may also associate with
microtubules of the mitotic spindle and regulate apoptosis during cell
cycle progression (32). While the staining patterns observed here may
be obscured due to the overexpression of the proteins, it does suggest
that Livin and Survivin have similar subcellular localization. If the
staining is an accurate reflection of endogenous Livin, it seems
possible that Livin could play a role in preventing caspase cleavage of both cytoskeletal and nuclear proteins during apoptosis and perhaps is
regulated by cell cycle proteins similar to Survivin.
To determine the critical motifs required for the localization of
Livin, HeLa cells were transfected with the deletion mutants Antisense to Livin Induces Apoptosis--
Since depletion of
Survivin using antisense was shown to produce defects in cell division
as well as increased caspase activation and apoptosis (24, 30-33), we
were interested in testing whether similar effects could be seen for
Livin. An antisense construct was designed to include the entire coding
region of livin cloned in the antisense orientation. There
was no open reading frame in the antisense orientation of
livin and therefore the construct did not result in the
expression of new proteins. To verify that the antisense construct
could reduce Livin expression, we compared the levels of overexpressed
Livin protein in cells transfected with antisense to that of a control
vector. To prevent apoptosis the cells were maintained in the presence
of the general caspase inhibitor ZVAD-fmk. In both HeLa (Fig.
7A) and G361 (not shown) cells, transfection with the antisense construct abrogated the expression of Livin seen with the V5 antibody. In contrast, the antisense did not reduce the levels of Survivin (Fig. 7A),
suggesting that the construct has a specific effect on Livin, and not
on related IAP family members.
The effect of the antisense construct on cell viability was analyzed by
co-transfection with an empty pTracer vector producing the GFP marker.
Transfections were performed in HeLa and G361 cells, which normally
express livin, as well as SW480 cells, which do not have
detectable levels of livin mRNA (Fig. 2). In HeLa or
G361 cells the antisense decreased the percent of GFP-positive cells by
80-90% relative to the vector and sense controls (Fig. 7B). In contrast, transfection of the antisense into SW480
had little effect on GFP levels (Fig. 7B). These results
demonstrate an effect of livin antisense on viability which
correlated with livin expression.
To extend these results, apoptosis was evaluated in more detail by
measuring DNA fragmentation and caspase activation. DNA fragmentation
was assessed using the TUNEL assay. Transfection of the antisense
construct into G361 cells triggered an ~8% increase in TUNEL
staining (Fig. 7C). Typically, the transfection efficiency in these cells was 15-20% suggesting that about half of the
transfected cells were TUNEL-positive. To test whether this correlated
with an increase in caspase activity, cytosolic extracts of the
transfected cells were incubated with the fluorescent substrate,
DEVD-AFC. Transfections with the antisense triggered nearly a 3-fold
increase in DEVD-AFC cleavage (Fig. 7D). Presumably, this
increase in caspase activity is a consequence of diminished
livin-caspase interaction.
These studies, along with those of Survivin, demonstrate significant
roles for these IAP family members in maintaining viability. While the
antisense to livin may act only on certain cell lines, it is
interesting that at least in HeLa cells, apoptosis resulted from
antisense to either livin or survivin. On the
other hand, antisense against xiap had no effect on the
viability of HeLa cells (data not shown). Furthermore, expression of
survivin could not rescue from apoptosis triggered by
livin antisense and vice versa. This suggests that Livin and
Survivin are not redundant, with distinct and important roles in
regulating apoptosis.
. In vitro
binding studies demonstrated a direct interaction between Livin and the
active form of the downstream caspases, caspase-3 and -7, that was
dependent on the BIR domain of Livin. In addition, the unprocessed and
cleaved forms of caspase-9 co-immunoprecipitated with Livin in
vivo, and recombinant Livin could inhibit the activation of
caspase-9 induced by Apaf-1, cytochrome c, and dATP. The
subcellular distribution of the transfected Livin was analyzed by
immunofluorescence. Both Livin and Survivin were expressed in the
nucleus and in a filamentous pattern throughout the cytoplasm. In
contrast to the apoptotic activity, the COOH-terminal RING domain
mediated its subcellular localization patterning. Further studies found
that transfection of an antisense construct against livin
could trigger apoptosis specifically in cell lines expressing
livin mRNA. This was associated with an increase in DNA
fragmentation and in DEVD-like caspase activity. Thus, disruption of
Livin may provide a strategy to induce apoptosis in certain cancer cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(TNF-
)/Fas apoptotic signaling pathways (1-3). While first
identified in baculovirus, the IAP family has been conserved
evolutionarily from viruses to nematodes, flies, and several mammalian
species. There are currently five human IAP family members, c-IAP1,
c-IAP2, XIAP, NAIP, and Survivin (4-7). All of the human IAP family
members, with the exception of NAIP, have been shown to interact with
specific cysteine proteases, or caspases, required for the cleavage of certain proteins involved in the disassembly of the cell during apoptosis (8). The caspases are synthesized as inactive zymogen forms
which upon apoptotic stimulation are proteolytically processed in a
sequential manner into their active heterotetrameric forms. c-IAP1,
c-IAP2, XIAP, and Survivin have been reported to bind to and inhibit
the active forms of the terminal caspases-3 and -7, but do not interact
with caspases-8, which is the most proximal caspase from the
TNF-
/Fas receptor (9-12). However, in the case of Survivin, caspase
inhibition may be more indirect through association with Cdk4 leading
to inhibition of pro-caspase-3 by p21 (13). c-IAP1, c-IAP2, and XIAP
also bind to the zymogen form of caspase-9 thereby preventing its
proteolytic processing as well as the processing of downstream
proteases, such as caspase-3, -6, and -7 (14). Abrogation of caspase
activity, a common downstream component of apoptosis, enables IAPs to
have widespread anti-apoptotic potential.
-mediated
NF-
B activation, thereby providing an additional mechanism for the
IAPs anti-apoptotic activity (19). Recent studies have also found that
c-IAP1 and XIAP have ubiquitin ligase activity which leads to their
degradation during apoptosis and that this activity is dependent on
their RING domain (20). However, it is unclear if this is a general feature of the RING domain in other IAP family members.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
N154,
C86, and 86-154) were generated by PCR and subcloned into
the KpnI/ApaI sites within pcDNA3.1/myc-His
(Invitrogen, Carlsbad, CA). FADD, Bax, and DR6, were subcloned by PCR
from Incyte clones 3334311, 2057308, and 2733717 and ligated into
pTracer-SV40 (Invitrogen, Carlsbad, CA) at
EcoRI/NotI, EcoRV/NotI, and
NotI/SpeI sites, respectively. Caspase-9 was
prepared by PCR from Incyte clone 3590462 and subcloned into
pcDNA3.1/V5/His-TOPO. Apaf-1 was cloned into pcDNA3.1/V5/His-TOPO from
a PCR product generated from a human testis cDNA library
(CLONTECH, Palo Alto, CA). The pTracer-SV40-RIP
plasmid was prepared by PCR from pMH-RIP (34) and ligated into the
KpnI/NotI sites in pTracer-SV40. The
constructions of pTracer-SV40-RIP3 and pZeoSV2-Bcl-xL were
described previously (34). pcDNA3.1-myc-survivin was provided by Dr.
Kevin Hudson (AstraZeneca Pharmaceuticals, Macclesfield,
Cheshire, UK). The nucleotide sequences of all of the clones were
confirmed by fluorescent terminator cycle sequencing using an automated
377 DNA sequencer (PerkinElmer Life Sciences and Applied Biosystems,
Foster City, CA).
-D-galactopyranoside for 4 h. The
bacterial pellet was resuspended in phosphate-buffered saline plus
lysozyme (1 mg/ml) and lysed by sonication. Lysates were incubated with
1% Triton X-100 for 30 min at 4 °C and then centrifuged to remove
the insoluble material. Supernatants were added to a
glutathione-Sepharose 4B column and the bound protein was eluted with
PreScission protease (Amersham Pharmacia Biotech, Piscataway, NJ)
thereby cleaving the glutathione S-transferase fusion.
and cycloheximide. Cell lysates were prepared in 1 ml of RIPA buffer (0.01 M sodium phosphate (pH 7.2), 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1%
Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS) supplemented with a protease inhibitor mixture tablet (Roche Molecular Biochemicals, Indianapolis, IN) which were spun at 20,000 × g for 30 min at 4 °C. The soluble supernatants were immunoprecipitated using
either a monoclonal antibody against V5 (Invitrogen, Carlsbad, CA)
followed by protein G-Sepharose or with a monoclonal antibody against
Myc conjugated to agarose (Santa Cruz Biotechnology, Santa Cruz, CA). The samples were washed three times in RIPA buffer, boilied in Laemmli
buffer, and resolved by SDS-PAGE. The gels were semi-dry blotted onto
nitrocelluose and probed with a monoclonal antibody against caspase-9
(PanVera Corp., Madison, WI). Immuno-complexes were detected by
enhanced chemiluminescence (ECL) according to the manufacturer's
specifications (Amersham Pharmacia Biotech).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and amino acids sequences of
livin. A, the BIR domain is indicated
in bold, and the RING domain is underlined. B,
homology between the BIR domains of Livin and other IAP family members
were determined using the Clustal algorithm from the MegAlign software
(DNASTAR). In cases where the IAPs had three BIR domains (c-IAP1,
c-IAP2, XIAP, and NAIP), the second BIR domain is represented due to
evidence from XIAP that the middle BIR has a more critical role in
suppressing apoptosis. Shading indicates identical residues
among family members.
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Fig. 2.
Expression of livin.
Northern hybridization were performed from multiple tissue blots
prepared from adult and fetal mRNA as well as from several cancer
cell lines. Each lane contains 2 µg of poly(A) RNA.
Blots were probed against livin and an actin
control. PBL, peripheral blood leukocytes.
/Fas pathway. Bax is a potent inducer of
apoptosis but may not have a direct role in TNF-
/Fas pathway (42).
HeLa cells were transfected with these apoptotic genes in the pTracer
vector containing a GFP marker for accessing viability. Transfection
with any of the pro-apoptotic genes led to roughly a 90% reduction in
viability, as compared with empty pTracer vector (Fig.
3A). Co-transfection of the
apoptotic genes with either livin or survivin
provided a 4-6-fold increase in viability. In general, the
anti-apoptotic activity of Livin appeared to be slightly more robust
than Survivin although that may be due to small differences in protein
expression. Levels of Livin and Survivin were monitored by Western
blotting and were comparable (data not shown). Survivin has been shown
previously to be less effective at inhibiting apoptosis than other IAP
family members (11). These results clearly demonstrate that Livin is a
novel member of the IAP family capable of inhibiting apoptosis by
several stimuli.
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Fig. 3.
Livin suppresses apoptosis by multiple
stimuli. A, HeLa cells (2 × 105) were
co-transfected with 1 µg of pTracer-SV40 derived plasmids containing
the apoptotic genes (dr6, fadd, rip,
rip3, bax) along with 1 µg of
pcDNA3.1-myc-survivin, pcDNA3.1/V5/His-TOPO-livin, or empty pcDNA3.1
vector. B, HeLa cells were co-transfected with 1 µg of
pTracer-SV40-RIP3, or empty pTracer-SV40 vector, and 1 µg of
pcDNA3.1/myc-His-livin- N154, -
C86, and -86-154, or empty
pcDNA3.1 vector. Twenty-four hours post-transfection GFP levels were
measured by laser scanning cytometry. At least 5000 cells were analyzed
for each transfection. The graphs indicate relative
viability which was calculated
based on the percent of GFP-positive cells as compared with the
control, empty pTracer-SV-40 with pcDNA3.1. Error bars
represent standard error of the mean, n = 3.
C86; amino acids 1-86), the central BIR
domain (86-154; amino acids 86-154), or the COOH-terminal RING domain
(
N154; amino acids 154-280) were tested for their ability to block
RIP3-mediated apoptosis. While the BIR domain is the critical region
for anti-apoptotic activity within the IAP family, there may be some
variations depending on the particular IAP and/or apoptotic stimulus.
For example, a single BIR domain from c-IAP1, c-IAP2, or XIAP is
sufficient for inhibiting caspase activity and etoposide-mediated
apoptosis (10, 16), whereas the BIR domain from Survivin is not
sufficient for inhibiting Taxol-induced apoptosis (32). In addition,
the baculoviral IAPs require both the BIR domain and the RING domain for their anti-apoptotic activity (18). Site-directed mutagenesis of
XIAP revealed that actually the region adjacent to the BIR domain may
play an important role in its activity (43). Here, it was found that
the BIR domain of Livin was sufficient for inhibiting RIP3-mediated
apoptosis, and was nearly as effective as the wild-type protein (Fig.
3B). The NH2- and COOH-terminal fragments could be detected by immunofluorescence but had no effect on apoptosis triggered by RIP3. Thus, the BIR domain of Livin appears to be the
critical motif involved in blocking apoptosis.
to evaluate interactions with activated caspase-9,
or left untreated to examine the unprocessed form. Cell lysates were
immunoprecipitated with antibodies against the epitope tags on Livin
(V5) and Survivin (Myc) and then probed by Western blot for caspase-9.
In the absence of TNF-
, Livin, but not Survivin, immunoprecipitated
with the unprocessed 45-kDa form of caspase-9 (Fig.
4A). Upon stimulation with
TNF-
, an increase in binding was observed between Livin and the
35-kDa cleaved form of caspase-9. Both Survivin and Livin were
expressed in the lysates (Fig. 4B). These data demonstrate
an association between Livin and pro-caspase-9 and a partially
activated form of caspase-9 containing the NH2-terminal
prodomain. No interaction of Livin was seen with the fully active 12- and 20-kDa subunits of caspase-9 using the Biacore, suggesting that the
prodomain is required for the interaction.
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Fig. 4.
Livin interacts with caspase-9 and inhibits
its processing. A, HeLa cells (1 × 106) either untransfected or transfected with 4 µg of
pcDNA3.1/V5/His-TOPO-livin or pcDNA3.1-myc-survivin were
treated with or without TNF- (1000 units/ml) and cycloheximide (30 µg/ml) for 2 h. Cell lysates were prepared in RIPA buffer and
immunoprecipitated with antibodies against V5 or Myc. The samples were
then resolved by SDS-PAGE, transferred onto nitrocellulose, and probed
with an antibody against the NH2-terminal of caspase-9. The
unprocessed 45-kDa and cleaved 35-kDa forms of caspase-9 recognized by
the antibody are indicated. Heavy and light
chains of immunoglobulin, denoted by asterisks, are
less abundant in precipitations with the Myc antibody since it was
conjugated to agarose. B, HeLa cells (1 × 106) were transfected with 4 µg of
pcDNA3.1/V5/His-TOPO-livin or pcDNA3.1-myc-survivin as above.
Cell lysates prepared in RIPA buffer were immunoprecipitated and
Western blotted with the indicated antibodies. C, in
vitro processing of caspase-9 was tested by incubating
[35S]methionine-labeled caspase-9 (2 µl) with in
vitro translated Apaf-1 (6 µl), cytochrome c (10 µM), dATP (1 mM), and recombinant Livin (50 µM) at 30 °C for 1 h. The samples were resolved
by SDS-PAGE and visualized with a PhosphorImager.
. TNF-
-induced apoptosis involves a
complex signaling response that includes activation of caspase-8 at the
receptor level leading to the processing of downstream caspases
including caspase-3 and -7 (45). Caspase activity was measured using
the fluorescent substrate DEVD-AFC, used to specifically detect
DEVD-cleaving caspases (caspase-3, -6, -7, -8, and -10). Cell lysates
were prepared from HeLa cells transfected with livin, the
anti-apoptotic gene bcl-xL, or an empty vector
which were subsequently treated with TNF-
and cycloheximide.
Treatment with TNF-
and cycloheximide for 5 h led to ~7-fold
increase in DEVD-AFC cleavage (Fig.
5A). However, cells
transfected with livin exhibited reduced caspase activity by
15-20%, consistent with the transfection efficiency of these cells.
Comparable levels of caspase inhibition were observed upon transfection
with bcl-xL. These results demonstrate the
ability of Livin to inhibit DEVD-like caspase activity in vivo, although Livin may not directly inhibit these caspases and it is possible that this suppression is simply the result of abrogation of caspase-9 processing upstream of caspase-3.
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Fig. 5.
Livin inhibits
TNF- -triggered DEVD-like caspase activity and
binds to active caspase-3 and-7 in vitro. HeLa
cells (2 × 105) were transfected with 1 µg of
pcDNA3.1/V5/His-TOPO-livin, pZeoSV2-Bcl-xL, or empty
pcDNA3.1 control. Twenty hours post-transfection the cells were
treated with TNF-
(1000 units/ml) and cycloheximide (30 µg/ml) for
5 h (hatched) or left untreated (solid).
DEVD-AFC hydrolysis produced from cytosolic extracts was monitored over
a 1-h period at 37 °C and quantified in relative fluorescence units
(RFU). Error bars represent standard error of the
mean, n = 3. B, equal amounts of recombinant
activated caspase-3 or -7 (1 µg) were incubated with
[35S]methionine-labeled Survivin, Livin, or the deletion
mutants,
C86 (NH2 terminus) and 86-154 (BIR domain),
which were prepared in vitro using the TNT T7 reticulocyte
lysate system. Samples were immunoprecipitated with antibodies against
the respective caspases and then resolved by SDS-PAGE and visualized by
autoradiography. Aliquots of the in vitro translated
(IVT) proteins (2 µl) were also analyzed in order to
determine their relative abundance.
C89 used as a negative control
(Fig. 5B). The BIR domain of Livin was sufficient for
caspase binding in agreement with its anti-apoptotic activity (Fig.
5B). A Biacore was used to determine the kinetics of the
interaction between Livin and the active forms of caspase-3, and -7. Relatively potent affinity constants were observed for both of the
caspases (24.6 pM for caspase-3 and 5.1 pM, for
caspase-7).
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Fig. 6.
Subcellular localization of
Livin. HeLa cells (2 × 105) were
transfected with 1 µg of pcDNA3.1-myc-survivin,
pcDNA3.1/V5/His-TOPO-livin, or pcDNA3.1/myc-His-livin N154.
Twenty-four hours post-transfection, cells were fixed and then
incubated with monoclonal antibodies against Myc or V5. Staining was
detected using a fluorescein isothiocyante-conjugated goat anti-mouse
antibody and visualized by epifluorescence, × 100 objective
lens.
C86, 86-154, and
N154 each containing a Myc epitope tag used for
staining. The truncated proteins
C86 and 86-154 produced aberrant
diffuse staining (not shown). However,
N154, which contains the
~35 amino acid RING domain, was found in the same filamentous pattern
as the wild-type protein (Fig. 6). These results suggest that the
COOH-terminal region of Livin, and perhaps more specifically the RING
domain, provides a sufficient signal for its proper subcellular distribution. While the function of RING domains have been largely enigmatic, they are often associated with mediating multiprotein complexes and in some cases co-precipitate with cytoskeletal proteins (47). Taking the functional and localization studies of Livin together,
one can propose a modular model whereby the BIR domain mediates its
anti-apoptotic activity and interaction with caspases, while the RING
domain provides proper cellular localization. Interestingly, other IAP
family members that have RING domains do not localize in the
filamentous pattern seen here (18). This disparity suggests that the
function of the RING domain, like the BIR domain, is not absolutely
conserved but rather may depend on either the particular IAP and/or
cell type in which it is expressed.
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Fig. 7.
Induction of apoptosis by livin
antisense. A, HeLa cells (2 × 105) were co-transfected with 1.5 µg of
pcDNA3.1/V5/His-TOPO-livin antisense, or control pcDNA3.1 vector, with
0.5 µg of pcDNA3.1/V5/His-TOPO-livin, pcDNA3.1-myc-survivin, or
control. Cells were maintained in 100 µM ZVAD-fmk to
prevent apoptosis. Cell lysates were prepared 24 h
post-transfection and probed with monoclonal antibodies against the V5
epitope (Livin), Survivin, and glyceraldehyde-3-phos- phate dehydrogenase (GAPDH) (control) and visualized
by ECL. B, HeLa, G361, and SW480 cells were co-transfected
with 0.5 µg of the pTracer-SV-40 marker plasmid along with 1.5 µg
of pcDNA3.1/V5/His-TOPO-livin (sense or antisense). GFP levels were
measured by laser scanning cytometry 48 h post-transfection. At
least 5000 cells were analyzed for each transfection. The graph
indicates relative viability which was calculated based on the percent
of GFP-positive cells as compared with the control, empty pTracer-SV-40
with pcDNA3.1. Error bars represent standard error of the
mean, n = 3. C, G361 cells were transfected
with 1.5 µg of pcDNA3.1/V5/His-TOPO-livin antisense, or control
pcDNA3.1 vector. Cells were harvested 24 h post-transfection and
DNA strand breaks were measured in situ using the TUNEL
procedure with fluorescein isothiocyante-labeled dUTP. The percent of
TUNEL-positive cells were assessed by laser scanning cytometry. At
least 2000 cells were counted for each sample. Error bars
indicate standard error of the mean, n = 3. D, HeLa cells were transfected with 1.5 µg of
pcDNA3.1/V5/His-TOPO-livin (sense or antisense), or control pcDNA3.1
vector. DEVD-AFC hydrolysis was measured in cytosolic extracts prepared
24 h post-transfection. AFC levels were monitored over a 1-h
period at 37 °C and recorded in relative fluorescence units
(RFU). Error bars indicate standard error of the
mean, n = 3.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. S. Hubbs for assistance with Biacore binding, L. Barnhart, M. Crickmore, and C. Schwarzman for generation of the caspase-9 and Apaf-1 constructs, and L. Hirata and P. Sherman for DNA sequencing.
![]() |
FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed: AstraZeneca
Pharmaceuticals, Enabling Science and Technology, CRDL 145, 1800 Concord Pike, Wilmington, DE 19803. Tel.: 302-886-8043; Fax:
302-886-8830; E-mail: bruce.gomes@astrazeneca.com.
Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M003670200
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ABBREVIATIONS |
---|
The abbreviations used are:
IAP, inhibitor of
apoptosis protein;
BIR, baculoviral IAP repeat;
TNF-, tumor necrosis
factor-
;
PCR, polymerase chain reaction;
MOPS, 4-morpholinepropanesulfonic acid;
GFP, green fluorescent protein;
AFC, 7-amino-4-trifluoromethylcoumarin;
PAGE, polyacrylamide gel
electrophoresis.
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