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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1435-1442
NEOPLASIA
Three differentially expressed survivin cDNA variants
encode proteins with distinct antiapoptotic functions
Edward M. Conway,
Saskia Pollefeyt,
Jan Cornelissen,
Inky DeBaere,
Marta Steiner-Mosonyi,
Kelly Ong,
Mathijs Baens,
Désiré Collen, and
Andre C. Schuh
From the Departments of Medicine and the Institute of Medical
Science, University of Toronto, Toronto, Canada; Center for Transgene
Technology and Gene Therapy; Department of Human Genetics, Flanders
Interuniversity Institute for Biotechnology, University of Leuven,
Leuven, Belgium.
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Abstract |
Survivin is a member of the inhibitor of apoptosis protein (IAP)
family that is believed to play a role in oncogenesis. To elucidate
further its physiologic role(s), we have characterized the murine
survivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons.
Surprisingly, 3 full-length murine survivin cDNA clones were
isolated, predicting the existence of 3 distinct survivin proteins. The
longest open reading frame, derived from all 4 exons, predicts a
140-amino acid residue protein, survivin140, similar to
human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second
cDNA, which retains intron 3, predicts the existence of a 121-amino
acid protein, survivin121 that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative
pre-messenger RNA (mRNA) splicing results in a third 40-amino acid
residue protein, survivin40, lacking the IAP repeat and
coiled-coil structure. Predictably, only recombinant
survivin140 and survivin121 inhibited caspase-3
activity. All 3 mRNA species were variably expressed during development
from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and
testis accumulated high levels of survivin140 mRNA, whereas
survivin121-specific transcripts were detected in all
tissues, while those representing survivin40 were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that
survivin plays a complex role in regulating apoptosis.
(Blood. 2000;95:1435-1442)
© 2000 by The American Society of Hematology.
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Introduction |
A delicate balance between cell proliferation and cell
death is required by multicellular organisms to maintain cell and
tissue homeostasis and thereby prevent the development of a variety of pathologic outcomes, including hematologic malignancies, hematopoietic dysfunction, cancer and lymphoproliferative diseases, autoimmune and
degenerative disorders, impaired wound repair, and developmental abnormalities.1,2 Apoptosis is the major physiologic means by which cell death is achieved. Although the cellular mechanisms responsible for mediating and regulating "programmed cell death" are not yet fully understood, it is clear that the process is complex
and tightly regulated. Current evidence supports a model in which the
induction of apoptotic cell death requires the activation of a series
of cysteine proteases, the caspases. After their release and activation
in response to a variety of stimuli, caspases lead to the destruction
of the cell in an apparently stepwise process, by disrupting the
nuclear membrane and chromatin structure, reorganizing the
cytoskeleton, shutting down DNA replication and repair, destroying DNA,
inducing phagocytosis, and finally disintegrating the cell into
apoptotic bodies.3 The complexity of this system is
underscored by recent data indicating that there also exist
caspase-independent pathways of apoptosis.4
Physiologic mechanisms to regulate apoptosis, and thereby prevent
uncontrolled cell destruction, are highly conserved through evolution.
The prototypical members of the inhibitor of apoptosis (IAP) family
were first identified in baculoviruses as proteins that could inhibit
the apoptotic response of cultured insect cells to viral
infections.5,6 Since that time, several additional members
of this family have been identified. The human XIAP, cIAP-1, and cIAP-2
proteins each contain 3 amino-terminal tandem repeats of the so-called
baculovirus repeat (BIR) domain, and a carboxy-terminal RING zinc
finger domain. The neuronal apoptosis inhibitor protein (NAIP),
however, contains no RING finger, whereas the "BIR repeat containing
ubiquitin-conjugating enzyme" (BRUCE) contains only a single BIR
repeat and lacks a RING finger domain.7 Although exhaustive
structure-function studies remain to be performed, it appears that the
BIR domain in these proteins is critical for antiapoptotic function,
although each individual BIR repeat may vary in specific
activity.8 Furthermore, the mechanism(s) by which IAPs
interfere with apoptosis remains unclear. Although XIAP, cIAP-1, and
cIAP-2 are able to inhibit caspase-3 and capase-7, NAIP is not,
consistent with the notion that additional caspase-independent pathways
of apoptosis may exist.9,10
Survivin is a recently described member of the IAP
family.11 It resembles BRUCE in that it contains only a
single BIR repeat and lacks a carboxy-terminal RING finger domain. In
addition, both survivin and BRUCE contain an insertion of 3 amino acid
residues within the BIR domain, which is absent in other IAP family
members.7 The gene encoding human survivin was
first identified by screening a human genomic DNA library with a
complementary DNA (cDNA) putatively encoding the coagulation factor Xa
receptor, effector cell protease receptor-1 (EPR-1).12,13
Because the coding regions of human survivin demonstrate a high
degree of sequence similarity with the corresponding regions of
EPR-1 in the antisense orientation, it was suggested that the 2 genes may be related evolutionarily11 and that the
regulation of the 2 genes may somehow be linked.14 The
functions of EPR-1 and survivin are not clearly related, however. EPR-1
is reported to be expressed widely in adult tissues and is believed to
mediate the proinflammatory or mitogenic properties of factor
Xa.15-18 In contrast, highest levels of steady-state survivin messenger RNA (mRNA) expression are found in solid
tumors and during fetal development, with much lower levels found in most adult tissues. Recent studies reveal that the expression of human
survivin is cell-cycle regulated and that its antiapoptotic function is
mediated both by its BIR domain and by the interaction of its
C-terminal coiled-coil domain with microtubules of the mitotic
spindle.14,19,20 The cDNA encoding the murine counterpart of human survivin has recently been reported.21
To elucidate the role(s) of survivin in physiologic and pathologic
conditions, before and after development, we characterized the murine
survivin gene and cDNA. We report that there exist 3 distinct
survivin cDNAs that are expressed differentially during development and
in adults, and that encode functionally distinct proteins. In addition,
we show that 3 distinct forms of survivin are also found in human
tissues and that these human variants are expressed differentially as well.
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Materials and methods |
Reagents
DNA restriction enzymes were obtained from Boehringer Mannheim
Canada (Dorval, Quebec) or New England Biolabs (Mississauga, Ontario),
and radioisotopes were from ICN Biomedicals (Mississauga, Ontario).
Murine 14.5 days postcoitum (dpc) embryo  phage cDNA and 129SV
genomic DNA libraries were purchased from Clontech (Palo Alto, CA) and
from Stratagene Cloning Systems (San Diego, CA), respectively. The
murine PAC library was provided by Genome Systems (South Bend, IN).
Recombinant human caspase-3 (catalogue no. 235 417) and the caspase-3
substrate colorimetric kit (catalogue no. 235 400) were purchased from
EuroBiochem (Bierges, Belgium). The caspase-3 has a specific activity
of approximately 1 U/mg protein, defined by the amount of enzyme that
cleaves 1.0 µmol of the substrate Ac-DEVD-pNA per minute at 25°C.
A panel of cDNAs derived from normal human tissues was obtained from
Clontech Multiple Tissue cDNA Panel 1, catalogue no. K1420-1.
Identification of cDNA and genomic DNA for murine survivin
Based on the published human EPR-1 cDNA sequence, a murine
expressed sequence tag (EST mc61a11.r1) was identified by BLAST search.
Identification of the cDNA encoding human
survivin12,13 revealed that this EST actually
corresponded to the murine homologue of survivin. Indeed, the
predicted amino acid sequence corresponding to this EST exhibited 86%
identity with the reported human survivin sequence, between amino acid
residues 1 and 128. EST mc61a11.r1 was subsequently purchased from
Genome Systems, the DNA sequence was confirmed, and the insert was
radiolabeled with  -32P-dCTP using the random primer
synthesis method22 for use as a probe to screen both the
murine embryo cDNA and genomic DNA libraries.
The phage cDNA library was prepared for plaque hybridization.
Filters were hybridized in Quik-Hyb (Stratagene) for 1 hour at 65°C
with the -32P-dCTP-labeled EST probe and finally washed
at 55°C with 0.5 × standard sodium citrate (SSC), 0.1%
sodium dodecyl sulfate (SDS). After 4 rounds of plaque purification,
the cDNA inserts were isolated and subcloned into pBS KS(+)
(Stratagene). The genomic DNA library was screened using the same
-32P-dCTP-labeled probe, and a single clone,
SRV10', was isolated, containing the entire coding region of
murine survivin. The ~13 kb insert was purified from the phage and inserted into the Not1 site of pBS KS(+). A murine
PAC library (Genome Systems) was also screened using the entire 13 kb
SRV10' insert as a probe. Three independent PAC clones were
identified and were shown by Southern blotting with gene-specific
radiolabeled oligonucleotide probes to contain the entire SRV10' sequence.
Fluorescent in situ hybridization
Fluorescent in situ hybridization (FISH) analysis was performed
following established procedures, by seeDNA Biotech Inc. (Toronto, Ontario) using the murine PAC survivin genomic clone as a
probe.23,24 Lymphocytes were isolated from mouse spleen and
cultured at 37°C in RPMI 1640 medium supplemented with 15% fetal
calf serum, 3 µg/mL concanavalin A, 10 µg/mL lipopolysaccharide and
5 × 10 5 mol/L mercaptoethanol. After 44 hours, the cultured lymphocytes were treated with 0.18 mg/mL BrdU for
an additional 14 hours. The synchronized cells were washed and
recultured for 4 hours in -minimum essential medium (MEM) with
thymidine 2.5 µg/mL. Chromosome slides were made as
reported.23,24 For FISH detection, the slides were heated
at 55°C for 1 hour, treated with RNAse A, and denatured in 70%
formamide in 2 × SSC for 2 minutes at 70°C, followed by
dehydration with ethanol. The biotinylated probe was denatured at
75°C for 5 minutes in 50% formamide and 10% dextran sulfate, and
hybridized overnight on the denatured slides. FISH signals and the DAPI
banding patterns were recorded separately and photographed. The FISH
signals were subsequently mapped to specific chromosomal bands by the
superimposition of FISH and DAPI images.
RNA isolation, Northern analysis, and reverse
transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was isolated from confluent cell monolayers or from
tissues by the method of Chomczynski and Sacchi.25 For
adult tissue samples, adult mice (10-12 weeks) were killed and tissues were dissected and placed into liquid nitrogen. For embryonic samples,
C57Bl/6 mice were mated, conception was assessed by the presence of a
coital plug (the morning of coital plug being scored as 0.5 days
postcoitum [dpc]), pregnant females were killed at various
developmental time points, and embryos were carefully removed by
dissection. Total RNA was extracted from adult and embryonic samples by
homogenization of the tissues, and cDNA was synthesized by reverse
transcription using murine leukemia virus (M-MLV) reverse transcriptase
and a cDNA synthesis kit (Bethesda Research Laboratories, Burlington,
Ontario). First-strand synthesis was primed using random
hexanucleotides, and PCR was performed as reported,26 with
the annealing temperature adjusted to 55°C. Pairs of
oligonucleotide primers for PCR were synthesized such that they flanked
the alternatively spliced mRNA forms. Primer srv86 (sense
5'-TCGCCACCTTCAAGAACTGGCCCTTCCTGGA), when paired with primer
srvas311 (antisense 5'-GTTTCAAGAATTCACTGACGGTTAGTTCTT) was
expected to generate PCR amplicons of 225 bp or 144 bp, depending on
the mRNA splice form. Primer srv86, when paired with srvas6380 (antisense 5'-GGCTTCTGACAATGCTTG), was expected to generate a PCR
amplicon of 332 bp. Resultant RT-PCR products were subsequently examined by agarose gel electrophoresis and by Southern blotting, using
a radiolabeled oligonucleotide probe, srv295 (sense
5'-TGGCTGCGCCTTCCTCACTGT) corresponding to the sequence found
within both amplicons. The amount of cDNA synthesized from each sample
was calibrated according to the relative expression of hypoxanthine
phosphoribosyl transferase (HPRT), as determined by agarose
gel electrophoresis of RT-PCR products generated using the specific
oligonucleotide primers, HPRT sense (5'-CACGGACTAGAACACCTGC) and
HPRT antisense, (5'-GCTGGTGAAAAGGACCTCT), yielding a 221 bp
product.27
Nested RT-PCR of RNA derived from human cell lines or tissue was used
to examine expression of human survivin transcripts.11 For
the same purpose, cDNAs derived from the mRNA of several normal human
tissues were purchased from Clontech . To determine whether a cDNA
lacking exon 2 was present, primers in exons 1 and 3 were paired. Thus,
primer hsrv2841 (sense 5'-CAGCCCTTTCTCAAGGAC) was first paired
with hsrv5175 (antisense 5'-GGAAAGCGCAACCGGACGAAT), and 2% of
the PCR product was subjected to a further 30 cycles of PCR using the
primer pair hsrv2861 (sense 5'-CCGCATCTCTACATTCAAGAAC) and
hsrv5159 (antisense 5'-CGAATGCTTTTTATGTTCCTC), yielding an expected product of either 193 bp or 83 bp, the latter expected if exon
2 is excised by alternative mRNA splicing. Nested RT-PCR was also used
to determine whether the homologue of murine survivin121 is
present in human-derived tissue or cells. Primer hsrv2841 was paired
first with primer hsrv5451 (antisense 5'-GAGGCAGGAGAATCACTTG), and 2% of the resultant amplicon was reamplified using primers hsrv2861 and hsrv5431 (antisense 5'-GAGAGGCAGAGGTTGCAGTG),
yielding products of either 480 bp (retention of intron 3) or 370 bp
(excision of exon 2). Resultant RT-PCR products were examined by
agarose gel electrophoresis and by Southern blotting using a
radiolabeled oligonucleotide probe, hsrv2888 (sense
5'-CTTCTTGGAGGGCTGCGCCTGCAC) corresponding to sequence in exon 1, which is found within both amplicons. For further verification, DNA
sequencing of gel-purified amplicons was performed.
Southern hybridization
The RT-PCR products were separated by electrophoresis on a 2%
agarose gel. Oligonucleotide probes were end-labeled with
 32P-ATP. Following Southern transfer, Genescreen nylon
filters (NEN Life Science Products, Brussels, Belgium)
were hybridized with radiolabeled probe in Quik-Hyb for 1 hour, washed
with 0.1% SDS, 0.5 × SSC at 55°C, and exposed to Kodak
XAR-5 film at  70°C.
Expression of recombinant murine survivin
cDNAs corresponding to the 3 forms of murine survivin were subcloned
in-frame into the expression vector pSecTagA (Invitrogen), to produce
survivin fusion proteins bearing an N-terminal Ig  -chain leader
sequence and a C-terminal myc epitope/polyhistidine tag. The resultant
plasmid vectorssurv140/psec, surv121/psec,
and surv40/psecwere then expressed stably into COS-7
cells using zeocin selection, conditioned media were collected, and the
myc/His6-tagged proteins were purified by nickel-column
chromatography. Recombinant proteins were eluted with 250 mM imidazole,
0.3 mol/L NaCl in phosphate-buffered saline (PBS), and the presence of
the expected forms of survivin was confirmed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western
immunoblotting using murine monoclonal anti-myc antibodies, followed by
detection with horseradish peroxidase (HRP)-conjugated goat-anti-mouse
antibodies and visualization with the ECL Western blotting system
(Pharmacia-Amersham, Rosendaal, Netherlands). Protein
concentrations were determined using bovine serum albumin as a standard.
Lysates of transiently transfected cells were also evaluated for the
expression of recombinant survivin variants. PBS-washed cell pellets
were suspended in a buffer containing 25 mM Hepes pH 7.5, 5 mM
MgCl2, 5 mM EDTA, 5 mM dithiotreitol, exposed to 3 freeze-thaw cycles, and centrifuged at 16 000g for 30 minutes. The lysates were then divided into aliquots and frozen at
20°C for subsequent analysis.
In vitro analysis of apoptosis
The effect of recombinant murine survivins on caspase-3 activity was
evaluated by using a specific colorimetric assay kit (EuroBiochem;
catalogue no. 235 417) according to the manufacturer's instructions.
Briefly, purified recombinant myc/His-tagged survivin (20-50 nmoles/L)
was added to 30 ng (3 × 105 units) of human
recombinant caspase-3 (EuroBiochem) in assay buffer (0.1 mol/L NaCl, 50 mM Hepes, 10 mM DTT, 1 mM EDTA, 10% glycerol, 0.1% CHAPS
[3-([3-Cholamidopropyl] dimethylammoniol)-1-propane-sulfonate], pH
7.4) in a final volume of 120 µL. The reaction was started by
addition of 10 µL of the colorimetric caspase-3 substrate I and the
rate of change in absorbance at 405 nm was measured using a kinetic
enzyme-linked immunosorbent assay plate reader. Controls without either
caspase-3 or substrate resulted in no change in absorbance during 30 minutes of observation. Experiments were performed in triplicate,
repeated 3 times, and the means and standard errors were calculated.
Antibody production
Polyclonal antibodies against murine survivin were generated by
Eurogentec (Seraing, Belgium) using a standard immunization protocol
whereby rabbits were immunized with KLH-conjugated peptides NH2-ALPQIWQLYLKNYRI-COOH and
NH2-CFKELEGWEPDDNPI-CONH2, representing amino
acid residues 5 to 19 and 60 to 74, respectively, of the deduced murine
amino acid sequence. Immunoglobulin was purified from immune sera by
protein-A sepharose chromatography, and specific antisurvivin
antibodies (Ig-srv .336) were subsequently obtained by affinity
chromatography of the immunoglobulin fractions on EAH-sepharose 4B
columns (Pharmacia-Amersham Biotech) to which the survivin peptides had
been coupled.
Biotinylation of proteins, immunoprecipitation, and Western
blotting
Embryonic samples, 12.5 dpc, were pooled and homogenized in PBS with
5 mM EDTA, 5 mM MgCl2. The clear cell lysate, obtained following centrifugation at 14 000g at 4°C for 30 minutes,
was incubated with biotin (EZ-Link Sulfo-NHS-LC-LC-Biotin, Pierce, Polylab N.V., Antwerp) for 60 minutes at 4°C, after which the reaction was stopped by the addition of 0.1 mol/L Tris-HCl.
Biotinlyated proteins were separated from free biotin by size
fractionation on a PD-10 column (Pharmacia) with PBS as
the running buffer. The resultant biotinylated protein solution was
incubated in PBS with 0.5 mol/L NaCl with either 20 µg of Ig-srv336
or preimmune immunoglobulin for 2 hours, after which protein
A-sepharose was added for a further 1 hour. The immunoprecipitated
pellets were washed 3 times with PBS/0.5 mol/L NaCl and twice with PBS.
Samples were boiled in nonreducing Laemmli buffer and separated on an 18% SDS-polyacrylamide gel. After transfer, the nitrocellulose filters
were blocked for 2 hours in Blotto, incubated with HRP-conjugated streptavidin for 1 hour, and then washed. The Amersham ECL
Western-blotting system, followed by exposure of the blots to
Hyperfilm-ECL, was used to detect specific bands. Prestained standard
markers were used to determine the approximate molecular weight.
Analysis of data
Alignments of DNA and amino acid sequences were performed with the
aid of MacVector software (Oxford Molecular Ltd, Oxford, UK).
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Results |
Identification of the murine survivin gene and cDNAs
A murine survivin cDNA probe was identified by searching the
dbest database for a murine ESTclone mc61a11.r1that matched closely
the cDNA encoding human EPR-1.11 By FASTA
analysis,28 EST mc61a11.r1 exhibited 85% and 79% sequence
similarity to the corresponding portions of human survivin and
EPR-1, respectively. Using this EST as a probe, 1 genomic and 9 cDNA clones (ranging in size from 800 bp to 2.1 kb) were identified by
phage library screening. Each of the cDNA clones was sequenced in
its entirety on both strands, while approximately 11 kb of the ~13 kb
genomic clone was also sequenced. A murine genomic PAC DNA library was subsequently screened with the purified survivin genomic DNA
probe, resulting in the identification of 3 independent, overlapping PAC clones. A biotin-labeled PAC DNA clone containing the entire survivin coding region (see below) was then used for FISH to
localize the survivin gene to chromosome 11E2 (Figure
1). Restriction mapping and partial
sequencing of DNA fragments from the PAC clones confirmed that the
phage genomic clone and the PAC clones were overlapping and
represented a single gene (data not shown).
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| Fig 1.
FISH analysis for chromosomal localization of murine
survivin locus.
Lymphocytes were isolated from mouse spleen and cultured as detailed in
"Materials and Methods." A biotin-labeled PAC DNA clone
containing the entire survivin gene was hybridized with
metaphase lymphocyte chromosomes. FISH signals and the DAPI banding
pattern were recorded separately, and the assignment of FISH mapping
data with chromosomal bands was achieved by superimposing FISH signals
with DAPI-banded chromosomes. In the left panel, dots represent the
specific FISH signals detected on chromosome 11. The right panel shows
the same mitotic figure, stained with DAPI to identify mouse chromosome
11.
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Four murine survivin cDNAs (clones 5.25, 92.16, 41.1, and 42.5)
corresponded very closely to their full-length human counterparts. The
largest of these, clone 5.25, comprised 48 bp of 5' untranslated sequence (UTR) upstream of an ATG translation initiation codon, a 420 bp open reading frame (ORF), and 872 bp of 3' UTR following an
in-frame TAA stop codon, and predicted a 140 amino acid
proteinsurvivin140 of molecular weight 16 286 Da, with
84% sequence similarity to human survivin (Figure
2). Comparison of the DNA sequence of this full-length cDNA clone with that of the murine survivin phage and PAC genomic clones revealed that the structural organization of the murine survivin locus was very similar to that of its
human counterpart (Figures 3 and
4). The murine survivin gene
comprises 4 exons. Exon 1, containing the ATG translational start site
(position 2865), is preceded by a putative TATA-less promoter and a
GC-rich region extending approximately 250 nucleotides in the 5'
direction. Exons 1 to 4 (111, 110, 118, and 81 bp, respectively) are
separated by 3 introns 292, 2797, and 2448 nucleotides in length. Exon
4 contains an in-frame TAA stop codon at nucleotide position 8822.
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| Fig 2.
Comparison of human survivin and murine survivin.
The predicted amino acid sequences of murine survivin140
(derived from cDNA clones 5.25, 92.16, 41.1, and 42.5),
survivin121 (derived from cDNA clones 7HB and 7L .15), and
survivin40 (derived from cDNA clones 5.20, 91.1, and 5.24)
are aligned with the amino acid sequence of human survivin.
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| Fig 3.
Sequence of the murine survivin gene.
The phage genomic DNA clone containing all 4 exons of murine
survivin was sequenced on both strands. The 4 exons, with
flanking intron sequence, are shown. Each exon is numbered, and the DNA
encoding exons 1 to 4 are in uppercase letters. The predicted amino
acid sequence of survivin140, derived from exons 1 to 4 is
also shown. Survivin121 is predicted to be derived from
retention of intron 3, whereas survivin40 is derived from
pre-mRNA splice removal of exon 2. Underlined DNA sequence represents
the only region of murine survivin cDNA without homology to
putative cDNA of human EPR-1.
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| Fig 4.
Murine survivin gene and cDNAs.
Exons 1 to 4 are denoted in red, light green, blue, and green,
respectively. Intron 3 is gray, and the 3' flanking region is
pink. (A) Restriction map of murine survivin gene. The arrow
indicates the ATG translational start site. Restriction enzyme sites
are shown: K (Kpn1); Stu (Stu1); Spe (Spe1); B
(BglII); RV (EcoRV); A (Asc1); S
(Sac1); P (Pml1); Apa (Apa1); Bc
(Bcl1). Location of DNA probes used for Southern
blots are shown. (B) Exon organization of the 3 distinct cDNAs encoding
the predicted survivin proteins. The arrow indicates the start of
translation. The heavy black vertical line represents the end of the
coding region. Survivin140 is derived from all 4 exons.
Survivin40 is generated by removal of exon 2-derived
sequences, with a resultant in-frame stop codon, and a truncated
protein. Survivin121 is derived from exons 1 to 3, and a
short part of intron 3, the latter being presumably retained during
pre-mRNA processing.
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Although 9 independent survivin cDNA clones were isolated, only
4 corresponded to a full-length form comprising exons 1 to 4. The
remaining 5 clones corresponded to 2 variant cDNAs that appeared to
have arisen by alternative pre-mRNA splicing (see Figures 2 and 4).
Clones 5.20, 91.1, and 5.24 corresponded to a shorter transcript that
lacked exon 2-derived sequences, and therefore contained an ORF of only
120 nucleotides followed by a 3' UTR, predicting a
truncated 40 amino acid residue proteinsurvivin40with a
molecular weight of 4667 Da (see Figure 4). In contrast, clones 7HB and
7L .15 contained exons 1 to 3 and retained intron 3 as well.
Acquisition of a new in-frame stop codon within intron 3 results in an
ORF of 363 nt, predicting a 121 amino acid protein (survivin121) with a molecular weight of 14 172 Da
(see Figure 4).
Alignment of survivin protein sequences with those of other
BIR-containing proteins
The amino acid sequences of the 3 variant murine survivin proteins
were aligned (see Figure 2) and compared with those of other
BIR-containing IAP protein members (not shown). The amino-terminal 113 amino acid residues of survivin121 are identical to those of survivin140. The remaining carboxy-terminal portions of
these 2 variants are distinct. Amino acids 15 to 87 comprise the
cysteine/histidine-containing zinc-binding BIR domain of
survivin140 (and survivin121). This region
exhibits 30% to 40% sequence identity with each of the BIR-domains of
murine XIAP, NAIP, and BRUCE. In common with human survivin, NAIP, and
BRUCE, murine survivin does not contain a carboxy-terminal RING finger.
Survivin40 shares sequence with only the amino-terminal
30% of the BIR domains of survivin140 and
survivin121 and does not contain a region rich in cysteine or histidine residues, suggesting that it likely does not have direct
antiapoptotic function.
Tissue distribution and developmental expression of
murine survivin mRNAs
Using mRNA variant-specific RT-PCR, we determined the relative
distribution of the 3 survivin transcripts in a variety of murine tissues, including brain, heart, kidney, liver, lung, ovary, pancreas, spleen, testis, and thymus. As illustrated in Figure 5, survivin140-specific mRNA
was detectable only in thymus and testis. In contrast,
survivin121-specific transcripts were detectable in all
tissues, although the signal was strongest in brain and ovary.
Survivin40 mRNA, on the other hand, was not
detectable in any of these tissues.
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| Fig 5.
Tissue distribution of murine survivin mRNAs.
RT-PCR was performed on mRNA derived from adult tissues using primers
that flanked the specific survivin mRNAs. (A) Accumulation of mRNA for
survivin140 was detected in thymus and testis (primer pair
srv86 and srvas311 results in amplicon of 254 bp). Positive controls
using cDNA clones 5.20 and 5.25 (see Figure 2) as target DNA, are in 2 lanes at the right side, demonstrating prominent 144 bp and 254 bp
bands, respectively. (B) Survivin121 mRNA was detected in
all tissues (primer pair srv86 and srvas6380 results in amplicon of 332 bp). Survivin40 mRNA using primer pairs srv86 and srvas311,
or srv86 and srvas6380 (expecting amplicons of 144 bp or 222 bp,
respectively) was not detectable. (C) HPRT transcripts were
present in all tissues (221 bp). The right lane in panels B and C are
negative controls without target DNA.
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Previous studies have demonstrated that survivin is expressed
in mouse embryos as early as 11.5 dpc.19 We examined the
developmental expression of the 3 survivin mRNA variants by
RT-PCR (Figure 6). In contrast to what we
had observed in adult tissue, all 3 survivin mRNA species,
including those corresponding to survivin40, were readily
detectable throughout development from 7.5 dpc, the earliest time point
examined, until 14.5 dpc. In all cases, the identity of the amplicons
generated by RT-PCR was confirmed by Southern blotting using the
radiolabeled internal oligonucleotide srv295. In addition,
representative PCR products of each size were purified and subcloned,
and their identity was verified by DNA sequencing.
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| Fig 6.
Developmental expression of murine survivin mRNA.
RT-PCR was performed on mRNA derived from murine embryos at different
developmental time points (7.5-14.5 dpc) using oligonucleotide primers
as noted in Figure 5. (A and B) All 3 survivin mRNA species
were detected, although survivin140 mRNA (254 bp) was the
most prominent. (C) HPRT transcripts were present at all
developmental time points (221 bp). These results are representative of
experiments on 3 embryos at each time point.
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To confirm directly whether the different survivin transcripts detected
in mouse embryos resulted in translation of distinct protein products,
we isolated 12.5 dpc murine embryos and biotinylated the lysates for
immunoprecipitation with an antisurvivin immunoglobulin that
specifically identifies all 3 recombinant forms. We could detect bands
representing survivin140 and survivin121,
confirming that in embryos that are 12.5 dpc, at least 2 of the
transcripts are translated into survivin proteins of the predicted
sizes (Figure 7). The immunoprecipitation
did not yield a band with a predicted molecular weight consistent with
survivin40.
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| Fig 7.
Immunoprecipitation of murine survivin from embryos.
Lysates of 12.5 dpc embyros were biotinylated and immunoprecipitated
with anti-survivin antibodies (lane 1) or nonspecific antibodies (lane
2). Following separation by SDS-PAGE and transfer to nitrocellulose
membranes, filters were incubated with streptavidin-HRP for detection
with the ECL kit (Amersham), as detailed in "Materials and
Methods." Molecular weight markers are on the left. Bands
representing survivin140 and survivin121 are
visualized at approximately 16 kd and 14 kd, respectively.
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Interference of caspase-3 activity by recombinant murine survivin
forms
To test directly the function of the variant murine survivin
proteins, recombinant myc/His6-tagged survivin fusion
proteins were created. The cDNAs encoding the survivin forms were
subcloned into the expression vector pSecTagA, and the resultant
vectors surv140/psec, surv121/psec, and
surv40/psec cDNAs were each transfected stably into COS-7
cells. Western immunoblotting of cell lysates with anti-myc or
antisurvivin antibodies (Ig-srv336) revealed single bands of
appropriate molecular size for each of the survivin forms.
Following purification of recombinant myc/His-tagged survivin variant
forms from conditioned media, varying concentrations were tested in
caspase-3 activity assays. In a representative set of experiments
summarized in Table 1, both
survivin140 and survivin121 suppressed
exogenous caspase-3 activity with similar dose-response curves.
At submaximal concentrations, survivin140 and
survivin121 had an additive effect with respect to
inhibiting caspase-3 activity. As predicted, survivin40 had
no effect on caspase-3 activity, and furthermore, we did not observe
any effect of survivin40 on the caspase-3 inhibiting
function of survivin140 or survivin121.
Expression of survivin mRNA variants in human cells and tissues
Using the published genomic DNA sequence of human
survivin,11 we designed a series of
oligonucleotides that were expected to identify similar human
survivin cDNA variants by RT-PCR. Oligonucleotide primers
hsrv2841, hsrv2861, and hsrv2888 lie within exon 1, hsrv5159 and
hsrv5175 are within exon 3, whereas hsrv5431 and hsrv5451 lie within
intron 3. As noted in Figure 8A, mRNA
transcripts corresponding to full-length survivin were detected in all
cell lines surveyed, including those derived from patients with
leukemia, colon cancer, neuroblastoma, melanoma, hepatoma,
neurofibrosarcoma, and carcinoma. Transcripts were also detected in
mRNA from 4 patients with mucosa-associated lymphoid tissue-type (MALT)
lymphomas, as well as in normal human tonsil, lymphoid tissue, kidney,
pancreas, liver, brain, and lung (not shown). The identity of the
transcripts was confirmed by Southern blotting with an internal
oligonucleotide probe specific to survivin or EPR-1. As demonstrated in
Figure 8B, we readily detected survivin transcripts corresponding to
murine survivin121 in RNA from cell lines derived from 2 human leukemias, a germ cell tumor, a teratocarcinoma, melanoma,
hepatoma, and carcinoma, but not in a colon cancer, neurofibrosarcoma,
or in normal lymphoid tissue, tonsil, kidney, pancreas, brain, or lung.
Weak signals corresponding to this transcript were also found in all 4 MALT lymphoma samples (not appreciated in Figure 8). Transcripts
corresponding to the human counterpart of survivin40
were detected in RNA derived from a neuroblastoma cell line and from 1 of the MALT lymphomas.
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| Fig 8.
Expression of human survivin mRNA forms.
Prior to separation by agarose gel electrophoresis, nested RT-PCR was
performed on mRNA derived from the following human cell lines and
tissues: 1. K562 leukemia cell line; 2. Jurkat T-cell line; 3. germ-cell tumor derived cell line; 4. teratocarcinoma cell line; 5. neuroblastoma cell line; 6-9. malignant tumor from patients with MALT
lymphoma; 10. AS19 melanoma cell line; 11. Del leukemia cell line; 12. SW620 colon cancer cell line; 13. Hep3B hepatoma cell line; 14. Ht1080
neurofibrosarcoma cell line; 15. A431 carcinoma cell line; 16. Raji
leukemia cell line. (A) Ethidium bromide-stained agarose gel of nested
RT-PCR products generated with oligonucleotide primers hsrv2861 and
hsrv5431. Transcripts correspond to full-length murine
survivin140. (B) Southern blot using
32P-labeled hsrv2888 to detect specific nested RT-PCR
products generated using oligonucleotide primers hsrv2861 and hsrv5159.
Several cell lines express the specific transcript corresponding to
murine survivin121, whereas only the neuroblastoma cell
line expresses a transcript corresponding to murine
survivin40.
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Discussion |
The human survivin gene has been reported to be highly
similar to the reverse complement of the EPR-1
gene.11 Indeed, the human survivin gene was
originally identified by screening a genomic library with a human
EPR-1 cDNA. Several lines of evidence indicate that the murine
gene and cDNAs that we have isolated correspond to the murine homologue
of survivin, rather than of EPR-1. First, the human
EPR-1 gene comprises 5 exons, with the mature protein being
encoded by an ORF of 1014 nucleotides. In contrast, the human and
murine survivin genes contain 4 exons, and the mature proteins
are encoded by ORFs of 426 and 420 nucleotides, respectively. Second,
both the sizes and nucleotide sequences of the murine survivin
exons are similar to those of their human counterpart. Third, although
the reverse complement of the most 3' end of human EPR-1
exhibits sequence similarity to survivin, and the corresponding exonsalthough transcribed in opposite directionsdemonstrate considerable overlap, despite extensive DNA sequencing of our murine
survivin genomic clones, regions homologous to exon 1 or the
5' part of exon 2 of EPR-1 (those regions of
EPR-1 which are not similar to the reverse complement
of human survivin) could not be found. Finally, attempts to
transcribe the opposite strand of the murine survivin gene by
computer analysis did not reveal additional potential intron-exon
structures that could encode a protein similar to EPR-1. We are
confident, therefore, that we have isolated the murine homologue of
survivin, rather than of EPR-1. Furthermore, in view of
recent claims that survivin and EPR-1 may be members of
a closely linked family of genes,14 we tried to determine
whether the murine genome also contains a family of
survivin/EPR-1-related genes (data not shown). Southern blot
analyses of murine ES cell genomic DNA and a murine survivin PAC genomic DNA clone did not suggest the presence of more than a
single gene, nor did FISH analysis detect more than a single locus.
Nonetheless, these are negative results and do not exclude definitively
the possibility that a family of genes does exist.
In these studies, we report the isolation and characterization of 3 independent survivin mRNA species from a murine embryonic cDNA
library. Examination of the corresponding cDNA and genomic DNA
sequences indicated that each of these transcripts originates from the
same gene. The cDNA encoding murine survivin140 is derived from all 4 exons. The corresponding deduced amino acid sequence predicts a protein with striking similarity to full-length survivin of
human origin, containing a single amino-terminal BIR repeat of
approximately 70 amino acids, and lacking a RING finger domain, yet
containing a 40 residue coiled-coil carboxy-terminal domain. A second
cDNA, derived from the same survivin gene, but lacking exon
2-derived sequence, predicts the existence of a truncated 40 amino acid
proteinsurvivin40 that does not contain the BIR element
believed to be critical for antiapoptotic function. Finally, a third
cDNA, which retains intron 3, and thereby acquires a new in-frame stop
codon, predicts the existence of an additional 121 amino acid
proteinsurvivin121. Both survivin140 and
survivin121 contain an intact BIR domain, and as expected,
interfere with caspase-3 activity. In contrast to
survivin140, survivin121 does not contain the
tubulin-interacting carboxy-terminal coiled-coil region. Although we
have not yet demonstrated that survivin140 and
survivin121 differ in their ability to interact with
tubulin-containing structures, we anticipate that in the absence of the
coiled-coil domain, survivin121 activity will not be
restricted spatially in a cell cycle-dependent manner.
Consistent with previous reports for human
survivin,11 expression of murine
survivin140, as detected by RT-PCR, is most
prominent in proliferating adult tissues. In contrast, murine
survivin121 mRNA transcripts appear to be
more widely distributed, being found in most adult tissues surveyed, a
finding in keeping with the observations of Kobayashi et
al21 who also showed that murine survivin is present in
several adult tissues and all T-cell and B-cell lines. We were unable
to detect survivin40 mRNA transcripts in
any of the adult murine tissues sampled. The failure of Ambrosini et
al11 to detect human
survivin121-specific RNA by Northern blot
analysis likely indicates that the 3' UTR survivin probe
that they used lies beyond the region encompassed by this variant
transcript. This notion is borne out by our studies in which each of
the survivin mRNA forms is detectable by RT-PCR in a variety of
human cells and tissues. Examination of the human survivin
genomic DNA sequence11 predicts that the human counterparts
for murine survivin121 and survivin40 would be
128 (human survivin128) and 40 (human
survivin40) amino acid residues in length, respectively.
Human survivin128, similar to its murine counterpart, would
retain the BIR domain and lose its C-terminal coiled-coil structure.
Human survivin40 would lack both a BIR domain and the
coiled-coil structure. In several normal adult tissues, only
transcripts corresponding to full-length human survivin were detected.
In contrast, in addition to transcripts for full-length human survivin,
most human tumors and tumor cell lines examined also expressed
transcripts corresponding to survivin128, whereas a
neuroblastoma cell line and 1 of the MALT lymphomas contained
survivin40-specific transcripts as well. Based on our
observation of an apparent change in the pattern of transcription of
the variant survivin forms, it seems reasonable to assume that the
differential expression of 1 or more of the functionally distinct
survivin variants might affect malignant behavior of a tumor, and
thereby alter prognosis. A recent report attempted to correlate the
tumor-specific expression of survivin with prognosis in patients with
neuroblastoma.20 Notably, this study detected survivin
expression immunohistochemically using antibodies that would not be
expected to distinguish among the 3 survivin forms. Our studies did not
allow the roles of survivin variants in defining tumorigenicity to be
assessed. We believe, however, that a more extensive evaluation,
coupled with specific in situ localization of the survivin variants,
will provide further insights into the role of the survivin variants in
normal and abnormal cell growth and proliferation.
Based on the putative structure of human survivin, investigators
hypothesized that its function would be similar to that of other
members of the IAP family.11 The antiapoptotic function of
IAP-class proteins is believed to be partially mediated via their
BIR-domains. In Drosophila, several proteins, including the
so-called death proteins, reaper and doom, are known to interact with
IAP member proteins, thereby altering the binding of the BIR-containing
protein to caspases, and diminishing their antiapoptotic function.10,29,30 Both human and murine full-length
survivins have been shown to bind specifically to caspases 3 or 7 (or
both) via the BIR-domain and to inhibit apoptosis in in vitro
systems.21,31 Regulation of this interaction has been
further elucidated by recent studies in which survivin expression in
HeLa cells was shown to be cell-cycle dependent and largely restricted
to the G2/M phase, a period during which the C-terminal
coiled-coil structure of survivin binds to microtubules of the
mitotic spindle, thereby enhancing interaction with target
caspases.32 It has been hypothesized that the
overexpression of survivin observed in most human cancer cell
lines31 reflects, at least in part, escape from this
apoptotic checkpoint during mitosis.32
These new insights into the mechanisms by which survivin may regulate
the apoptotic pathway are particularly intriguing in light of our
finding that there exist 3 variant forms of survivin, each containing
distinct structural domains. Survivin140, similar to human
survivin, contains both a BIR-domain and a coiled-coil carboxy-terminal
region, and consequently would be expected to regulate apoptosis in a
cell cycle-dependent fashion. In contrast, murine
survivin121, and its human counterpart, human
survivin128, lacks the coiled-coil structure, but contains
a BIR-domain. We have further confirmed that both murine
survivin140 and murine survivin121 can directly
inhibit caspase-3 activity. Because survivin121 is likely
unable to interact with microtubules, one would not expect it to
regulate terminal caspases 3 and 7 in a localized manner that is
similarly coupled to the cell cycle. Thus, murine survivin140 and survivin121, despite their
similarity, may have quite different spectra of biologic activity. The
complexity of this system is further underscored by the existence of
survivin40, which lacks both a complete BIR domain and a
coiled-coil region. Therefore, it is reasonable to speculate that the
differential expression of these forms of survivin during the cell
cycle might affect the balance between cell proliferation and
programmed cell death.
We propose that the various forms of survivin differ in their ability
to interact with other proteins involved in the regulation of apoptosis
(or possibly to self-associate), and therefore, that the role of
survivin in apoptosis regulation may vary in a manner that is defined
by the spectrum of survivin proteins present, and consequently, by the
combinatorial protein-protein interactions that are permitted. In a
purified system, we were not able to detect any effect of
survivin40 on the inhibition of caspase-3 activity by
either survivin121 or survivin140. Despite
these preliminary findings, it is still conceivable that through
protein-protein interactions, survivin40, for example,
might inhibit and thus regulate the antiapoptotic function attributed
to survivin. It follows that alterations in expression of 1 or more of
the survivin variants may be predicted to result in a variety of
disease processes, either degenerative or proliferative. In that
respect, mutations in the NAIP gene have been linked to motor
neuron degeneration in one of the spinal muscular
atrophies,33 while genetic deregulation of human IAP1 has
been associated with the development of MALT lymphomas.34
The wide tissue distribution and early developmental expression of the
variant forms of survivin make it a likely candidate to be involved in
several disease processes. Elucidation of the site-specific function(s)
of these survivin variants may therefore provide further insights into
the regulation of the apoptotic pathway in a variety of physiologic and
pathologic conditions.
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Footnotes |
Submitted April 5, 1999; accepted October 22, 1999.
Supported in part by the Heart and Stroke Foundation of Ontario and the
Medical Research Council of Canada.
Reprints: Edward M. Conway, Center for Transgene Technology and
Gene Therapy KU Leuven Campus Gasthuisberg, O&N Herestraat 49, 9th
floor, B-3000 Leuven, Belgium; e-mail: ed.conway{at}med.kuleuven.ac.be.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction