[CANCER RESEARCH 59, 6097– 6102, December 15, 1999]

Advances in Brief

Survivin-DEx3 and Survivin-2B: Two Novel Splice Variants of the Apoptosis

Inhibitor Survivin with Different Antiapoptotic Properties1
Csaba Mahotka, Michael Wenzel, Erik Springer, Helmut E. Gabbert, and Claus D. Gerharz2
Institute of Pathology, Heinrich Heine University, D-40225 Duesseldorf, Germany

Abstract other. Some were reported to directly bind and inhibit intracellular
cysteine proteases (caspases) activated in apoptosis or, in the case of
Recently, a novel antiapoptosis gene, i.e., survivin, was identified as c-IAP1 and c-IAP2, to bind TRAF1 and TRAF2 (3).
a structurally unique member of the inhibitor of apoptosis protein
Recently, the gene for a novel member of the IAP family, survivin,
family. Survivin expression is turned off during fetal development and
not found in non-neoplastic adult human tissues but is again turned on
was found by hybridization screening of a human P1 genomic library
in the most common human cancers. The antiapoptotic properties of with the cDNA of EPR-1 (4). Survivin is structurally unique among
survivin might provide a significant growth advantage in tumors and mammalian IAPs, containing only a single BIR and lacking the
possibly also contribute to chemoresistance of cancer. Therefore, we COOH-terminal RING finger domain. Additionally, in contrast to
analyzed the expression of survivin in human renal cell carcinomas c-IAP1 and c-IAP2, survivin has no caspase recruitment domain,
(RCCs), known to be largely resistant to chemotherapy. Northern blot which is necessary for the interaction with TRAFs.
analysis and RT-PCR revealed survivin expression in newly established It was shown that survivin inhibits processing of procaspase-3 and
RCC cell lines (n 5 11) of all major histological types. Moreover, we procaspase-7 and specifically binds both active caspases in vitro (5).
identified two novel splice variants of survivin, lacking exon 3 (sur- Remarkably, caspase-3 has a key role in promoting the apoptosis
vivin-DEx3) or retaining a part of intron 2 as a cryptic exon (survivin-
signal. Thus, caspase-3 is responsible for the cleavage of poly(ADP-
2B). Both sequence alterations cause marked changes in the structure
of the corresponding proteins, including structural modifications of the
ribose) polymerase, PAK2, lamin, gelsolin, and fodrin, resulting in
baculovirus inhibitor of apoptosis protein repeat domain. The role of inhibition of DNA repair, formation of apoptotic bodies, nuclear
the novel isoforms in the regulation of apoptosis was assessed in membrane breakdown, and cytoplasmic shrinkage, all of which are
transfection experiments, showing conservation of antiapoptotic prop- hallmarks of apoptosis (6).
erties for survivin-DEx3 and a markedly reduced antiapoptotic poten- Initial studies using Northern blotting, in situ hybridization, and
tial for survivin-2B. In conclusion, our observations suggest a complex immunohistochemistry revealed strong survivin expression in several
regulatory balance between the different isoforms of survivin, which fetal tissues, whereas no survivin transcripts were detected in a variety
might determine the response to proapoptotic stimuli, not only in of adult tissues (7). Most strikingly, survivin was observed to be
human RCCs but also in fetal tissues and other types of cancer. expressed in the most common human cancers, including carcinomas
Introduction of lung, stomach, colon, breast, and prostate as well as high-grade
non-Hodgkin’s lymphomas (4). These findings suggested that cancer
Apoptosis plays an important physiological role during embryonic cells return to a fetal pattern of survivin expression to enhance cell
development and in the maintenance of tissue homeostasis. Deregu- viability and thereby possibly also become able to overcome the
lation of apoptosis has been implicated in carcinogenesis by abnor- cytotoxic effects of chemotherapeutic agents. In fact, immunohisto-
mally prolonging cell survival, facilitating the accumulation of trans- chemical studies in neuroblastomas and colorectal carcinomas indi-
forming mutations, and promoting resistance to immunosurveillance. cated a correlation between the expression of survivin and a clinically
Moreover, impairment of apoptotic cell death might significantly unfavorable course of disease, proposing survivin expression as a
affect resistance of cancer to chemotherapy and irradiation (1). potential prognostic factor (8, 9).
The molecular pathways leading to apoptotic cell death are highly To analyze the expression of survivin in cell lines derived from
conserved evolutionarily and controlled by proteins that either pro- human RCC of all major histological types, we used a survivin-
mote or counteract apoptotic cell death. Regulators of apoptotic cell specific RT-PCR procedure, allowing the exclusive amplification of
death identified thus far predominantly include proteins of the Bcl-2 the entire survivin coding sequence. In this report, we describe the
family and the IAP3 family. First described as baculoviral genes, identification of two novel alternatively processed survivin tran-
several cellular homologues of IAPs have been identified in eu- scripts, designated as survivin-DEx3 (lacking exon 3) and survivin-2B
karyotes, all of which are characterized by two or three BIRs, a (retaining a part of intron 2 as a cryptic exon). The expression of both
COOH-terminal RING finger domain, and a caspase recruitment survivin and survivin-DEx3 was observed in all RCC cell lines tested,
domain (2). All mammalian IAPs suppress apoptosis, but their way of with most RCCs additionally expressing survivin-2B. The function of
function is not yet fully understood and could be different from each the novel survivin isoforms was assessed in transfection experiments,
showing a largely preserved antiapoptotic function for survivin-DEx3
Received 5/12/99; accepted 10/29/99. and a marked reduction of antiapoptotic potential for survivin-2B. Our
The costs of publication of this article were defrayed in part by the payment of page data, therefore, suggest that survivin variants generated by alternative
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact. splicing in human RCCs are intrinsically involved in the complex
1
This work was supported by the Mildred Scheel Stiftung. regulation of apoptosis.
2
To whom requests for reprints should be addressed, at Institute of Pathology,
Heinrich Heine University, Moorenstr. 5, D-40225 Duesseldorf, Germany. Phone: 49- Materials and Methods
211-811-8348; Fax: 49-211-811-8353.
3
The abbreviations used are: IAP, inhibitor of apoptosis protein; BIR, baculovirus IAP
Cell Lines and Cultures. All cell lines used in this study were derived
repeat; TRAF, tumor necrosis factor receptor-associated factor; EPR, effector cell prote-
ase receptor; RCC, renal cell carcinoma; RT, reverse transcription; EGFP, enhanced green from typical representatives of the clear cell, chromophilic/papillary, and
fluorescent protein; SD, splice donor; SA, splice acceptor. chromophobe types of RCC, established in our laboratory as described previ-
6097
 NOVEL SPLICE VARIANTS OF SURVIVIN

ously (10). The cell lines were maintained with DMEM (Life Technologies, 1 3 105 cells were plated into 24-well plates and incubated with 100 mg/ml of
Inc., Eggenstein, Germany) supplemented with 10% FCS, penicillin, and the potent chemotherapeutic drug methotrexate. HepG2 cells had been shown
streptomycin and cultivated at 37°C in an atmosphere with 5% (v/v) CO2. previously to be methotrexate responsive, exhibiting CD95-mediated apoptosis
Short-term cultures of nonneoplastic renal tubule epithelia were derived from upon exposure to this anticancer drug (12). Cell viability was measured after
renal cortical tissue samples obtained from nephrectomy specimens. The tissue 24-h incubation by scoring cells as alive or dead by trypan blue staining.
samples were mechanically macerated, and cultivation in standard growth Transfection experiments were performed at least three times.
medium resulted in rapid outgrowth of epithelial cells. Fibroblastic contami- To determine transfection efficiency, control cells were fixed 48 h after
nation of short-term cultures could be removed by selective trypsinization, as transfection (2% formaldehyde/0.2% glutaraldehyde in PBS) and stained for
shown by immunocytochemical analysis demonstrating a pure population of b-galactosidase expression (0.1% 5-bromo-4-chloro-3-indolyl b-D-galacto-
cytokeratin-positive epithelial cells. side/5 mM potassium ferricyanide)/5 mM potassium ferrocyanide/2 mM MgCl2.
RNA Extraction and Northern Blot Analysis. Total RNA was extracted Immunoblotting. Protein-normalized aliquots of SDS extracts of HepG2
from cell lines by guanidinium thiocyanate treatment and centrifugation in 5.7 transfectants were electrophoresed on 15% SDS-polyacrylamide gels and
mol/l cesium chloride solution. Poly(A)1 RNA was isolated from total RNA transferred to Optitran BA-S85 nitrocellulose membranes (Schleicher &
via the Oligotex kit (Qiagen, Hilden, Germany). Northern blotting and hybrid- Schuell, Dassel, Germany). The membrane was blocked over night in washing
ization were performed as described previously (11). buffer [100 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.2% Tween 20] plus
cDNA Synthesis. RT reactions contained 2 mg of total RNA, 13 RT buffer 3% nonfat dry milk/1% BSA, incubated for 1 h at room temperature with 1
(Promega, Heidelberg, Germany), 25 mM of each deoxynucleotide triphos- mg/ml rabbit antihuman survivin antibody (kindly provided by Dr. Dario
phate, 10 pmol of sequence-specific RT primer (59-AGG AAC CTG CAG Altieri, Boyer Center for Molecular Medicine, Yale University School of
CTC AGA-39, corresponding to nucleotides 914 –931 of the survivin antisense Medicine, New Haven, CT) or mouse anti-GFP (Clontech), washed, and
strand) or 0.06 mg of random primer (Stratagene, Heidelberg, Germany), 20 incubated with a 1: 2000 dilution of horseradish peroxidase-linked donkey
units of RNAsin RNase inhibitor (Promega), and 5 units of AMV reverse antirabbit or sheep anti-mouse antibody (Amersham Pharmacia, Freiburg,
transcriptase (Promega) in a final volume of 30 ml. The specific RT reactions Germany) for 1 h at room temperature. After washing, binding of the primary
were incubated at 50°C and random RT reactions at 37°C for 1 h, respectively. antibody was revealed by incubation with Lumi-Light substrate (Roche, Mann-
PCR Assays. PCR amplifications were performed on a PTC-100 Program- heim, Germany). Equal loading was confirmed by a-tubulin detection with an
mable Thermal Controller (Biozym Diagnostic, Hess, Oldendorf, Germany). anti-a-tubulin antibody (Sigma-Aldrich, Deisenhofen, Germany).
Three ml of cDNA mixture were subjected to amplification in a 50-ml mixture
containing 2.5 units of Taq polymerase, 13 PCR buffer (both from Qiagen), Results
25 mM each of dATP, dCTP, dGTP, and dTTP, and 25 pmol each of 59 and 39
primer. PCR conditions were: initial denaturation for 2 min at 94°C, followed Expression of survivin and Two Novel Splice Variants in RCC
by 30 cycles of denaturation at 94°C for 30 s, annealing for 1 min at 62°C, Cell Lines. By Northern blot analysis, all RCC cell lines (n 5 11)
extension at 72°C for 1 min, and a final extension at 72°C for 5 min. The displayed survivin expression, irrespective of their histological types
primer pair used for amplification of the survivin entire coding region was:
(Fig. 1a). This observation was further confirmed by RT-PCR, dem-
forward primer, 59-GCA TGG GTG CCC CGA CGT TG-39 [corresponding to
position 48 – 67 of the survivin mRNA (GenBank accession NM 001168)]; and
onstrating the expected survivin amplification product of 431 bp, and
reverse primer, 59-GCT CCG GCC AGA GGC CTC AA-39 (position 475– to our surprise, two other bands of 500 and 329 bp, respectively (Fig.
494). Glyceraldehyde-3-phosphate dehydrogenase controls were performed 1b). These three survivin-specific amplification products were cloned
using forward primer 59-ACG GAT TTG GTC GTA TTG GGC G-39 and and sequenced. The largest band was identified as a survivin transcript
reverse primer 59-CTC CTG GAA GAT GGT GAT GG-39. retaining a 69-bp long part of intron 2 (corresponding to nucleotides
Sequence Analysis. Bands of interest were eluted from agarose gels using 4460 – 4528 of the published sequence of the survivin gene; GenBank
the QIAquick gel extraction kit (Qiagen), ligated into the pGEM-T-cloning accession no. U75285) as a novel exon, termed exon 2B (Fig. 1c). The
vector (Promega), and cloned in accordance to standard protocols. Plasmid 431-bp product was identified as regularly spliced survivin. The
DNA was recovered using the Plasmid Mini kit (Qiagen), cycle sequenced, and
smallest band was a survivin fragment of only 329 bp in length with
analyzed in a ABI Prism 310 sequencing apparatus (Applied Biosystems,
the 118-bp of exon 3 missing. In addition, formation of heteroduplices
Weiterstadt, Germany) using T7 or SP6 site-specific primers.
S1 Nuclease Analysis. S1 nuclease analysis was performed using the S1 occurred but did not influence the ratio between the bands of expected
nuclease assay kit (Ambion, Austin, TX) according to the manufacturer’s length. Additional amplification of genomic DNA showed no product,
protocol. The sequences of oligonucleotide probes corresponded to exon/exon confirming the presence of each splice variant on mRNA level only.
borders of the complementary strand [survivin-DEx3: position 231–270/389 – Short-term cultures of nonneoplastic kidney tubule epithelia showed
428; survivin-2B: position 3244 –3283/4460 – 4499 (according GenBank ac- no expression of any survivin isoform by RT-PCR (data not shown).
cession no. U75285)]. Probes were labeled with [g-32P]dATP by T4-polynu- The existence of the newly identified survivin splice variant survivin-
cleotide kinase (Life Technologies) and purified according to standard DEx3 was further proved by distinctive S1 nuclease analysis, dem-
protocols (1). Samples were analyzed on 8% denaturing polyacrylamide gels
onstrating the presence of the mRNAs for both regular survivin and
(Sequi-Gen GT Sequencing Cell; Bio-Rad Laboratories, Munich, Germany),
alternatively spliced survivin-DEx3 transcripts (Fig. 1d).
and dried gels were submitted to autoradiography.
Cloning of survivin Coding Sequences. To generate EGFP-tagged sur- Splice Donor and Acceptor Sites Adjacent to the Novel Exon 2B
vivin constructs, the coding sequences of the three survivin variants were PCR Are Suitable for Alternative Splicing. Computer analysis with the
amplified as described above using the following primer sets: 59-GTC GTC Signal program of the PC/GENE package revealed that the novel
GGT ACC ATG GGT GCC CCG ACG TTG-39 (sense); 59-CAG CAG GGA 69-bp exon in survivin-2B is flanked by SD and SA sites (Fig. 2a).
TCC ATC CAT GGC AGC CAG CTG CTC-39 (antisense) for survivin and These sites matched to the consensus sequences of common SD sites
survivin-2B, respectively; and 59-CAG CAG GGA TCC AGA CAT TGC TAA ({C/A}AGuGT{A/G}AGT) and SA sites ({T/C}11N{C/T}AGuG) (13,
GGG GCC CAC A-39 (antisense) for survivin-DEx3. PCR reactions were 14). Thus, it is a well-known fact that the GT bases of SD sites and the
purified using Microspin S-300 columns (Pharmacia Biotech Europe, Freiburg, AG bases of SA sites are necessary to perform splicing processes on
Germany), digested with KpnI and BamHI, ligated into the mammalian ex-
exon-intron and intron-exon boundaries.
pression vector pEGFP-N3 (Clontech, Heidelberg, Germany), and cloned
according to standard protocols.
The SA site at position 4445– 4460 of exon 2B reads as follows:
Transfection of Cultured Cells, Induction of Cell Death, and Assess- CCTTAATCCTTACAGT. The homology of this SA site to the SA
ment of Cell Viability. Eight mg of pEGFP-N3 vector control and survivin consensus sequence (Fig. 2b) seems to be sufficient in some RCC cell
constructs were each cotransfected with 2 mg of pCMVb into HepG2 hepa- lines only to permit alternative exon usage, whereas other RCC cell
toma cells according to the DEAE-dextran protocol. After culturing for 24 h, lines were unable to perform insertion of exon 2B. This observation
6098
 NOVEL SPLICE VARIANTS OF SURVIVIN

Fig. 1. Expression of different survivin isoforms in human RCC cell lines. a, Northern blot hybridization revealed survivin expression in all RCC cell lines, irrespective
of their histological subtypes (clear cell, chromophilic, and chromophobe types of RCC). Of note, a band of 1.3 kb indicated EPR-1 expression in clearCa-6. b, RT-PCR
confirmed the results of Northern analysis showing survivin expression in all RCC cell lines as well as two additional fragments different in length from the survivin band.
c, sequence analysis of the RT-PCR amplification products identified three different transcripts: The survivin transcript with four exons, survivin-2B with an additional exon
(exon 2B) inserted between the exons 2 and 3, and survivin-DEx3, showing a loss of exon 3 as well as a frame shift with extension of the reading frame into the open reading
frame of the 39 untranslated region (arrows, PCR primer positions; flags, SD and SA sites; black boxes, the novel exon 2B; shaded boxes, the frame shift in exon 4 and the
coding part of the former 39 untranslated region). d, S1 analysis of the survivin-DEx3 transcript revealed the protected fragments of regular survivin (40 nucleotides) and
alternatively spliced (80 nucleotides) survivin-DEx3 transcripts.

could indicate patient and/or tumor specificity of the corresponding followed by an additional frame shift in exon 4. In consequence, the
splicing factors. open reading frame of survivin-DEx3 ends in the 39 untranslated
The 39 boundary of exon 2B has a SD at position 4526 – 4534 region of survivin at position 581 and generates a novel 63 amino
(GAGGTCAGG). This SD site (Fig. 2b) matches sufficiently to the acids-long COOH-terminal tail in survivin-DEx3 coded by 87 nucle-
consensus site and is capable of linking exon 2B to the SA site of exon otides of exon 4 and 103 nucleotides of the original 39 untranslated
3. No splice products, however, were found directly linking exon 2B region. Because of this frame shift, a novel potential N-myristoylation
to exon 4. domain was generated at position 121.
In contrast, the SD site of exon 2 is suitable for linking exon 2 In summary, both splice variants exhibited truncation of the func-
directly to three different exons of the survivin gene, i.e., exon 2B tionally important BIR domain at the same position but differed in
(survivin-2B), exon 3 (survivin), and exon 4 (survivin-DEx3) in their COOH-terminal regions. Additional posttranslational modifica-
human RCC lines (Fig. 1c). In conclusion, our data convincingly tions, e.g., by N-glycosylation and/or N-myristoylation, could deter-
indicated that the newly identified survivin variants are transcript mine differential activity and/or intracellular location of the corre-
isoforms generated by alternative splicing of the survivin pre-mRNA. sponding proteins.
Differential Alterations of Protein Domains in the Novel sur- Differential Antiapoptotic Activity of the Novel survivin Splice
vivin Splice Variants. Computational domain analysis of the puta- Variants. Because RCCs are known to be largely resistant to anti-
tive proteins of survivin-2B and survivin-DEx3 revealed crucial con- cancer drug-induced apoptosis, we used another apoptosis-inducible
sequences of the alternative splicing processes (Fig. 3). The chief tumor model for further functional analysis of the different survivin
characteristic of survivin-2B was the modification of the essential BIR splice variants. Therefore, we selected the HepG2 tumor cell line,
domain, which was interrupted by insertion of exon 2B. The inclusion because this cell line had been shown previously to respond to
of exon 2B generated a novel potential N-glycosylation site at position methotrexate with marked CD95-mediated apoptosis (12).
84 and two novel potential N-myristoylation sites (positions 89 and Using HepG2 cells, transfection experiments revealed marked dif-
92). Inclusion of exon 2B, however, did not result in a frame shift, as ferences in the antiapoptotic activity of the novel survivin splice
became evident in survivin-DEx3. variants (Fig. 4). In accordance with its previously described anti-
Skipping of exon 3 in survivin-DEx3 resulted in a modification of apoptotic potential, transient transfection of HepG2 cells with survivin
the BIR domain at the same position as in survivin-2B but was resulted in a marked increase of cell survival after exposure to
6099
 NOVEL SPLICE VARIANTS OF SURVIVIN

Fig. 2. Analysis of potential SD and SA sites between exon 2

and 3. a, computer-aided analysis of SD/SA sites was performed
on the survivin genomic sequence to confirm the data obtained
from RT-PCR and DNA sequencing. p, positions of potential SD
and SA sites that correspond to the SD and SA sites used in the
newly discovered survivin-2B splice variant. As a result, a part of
the intron between exon 2 and 3 is retained as a cryptic exon,
termed exon 2B (see Fig. 1c). SD and SA sites were analyzed with
the Signal program of the PC/Gene Package Ver. 6.60, according
to the algorithm developed by Staden (13). b, comparison of the
SA and SD sites of exon 2B with the consensus sequences for SA
and SD sites.

methotrexate when compared with the empty vector control. Closely cancers, including carcinomas of the lung, stomach, colon, breast, pros-
corresponding cell survival was observed after methotrexate treatment tate, and high-grade non-Hodgkin’s lymphomas (4). Reexpression of
of HepG2 cells transfected with survivin-DEx3. In contrast, HepG2 survivin in cancer might provide important selective advantages by
cells transfected with survivin-2B showed a marked reduction of cell abnormally prolonging cell survival and promoting resistance to apopto-
survival after exposure to methotrexate (Fig. 4). sis induced by immunocompetent cells or anticancer drugs.
These data strongly suggested differential antiapoptotic properties The biochemical mechanisms by which survivin suppresses apop-
of the novel survivin splice variants, showing a nearly complete tosis are still under investigation. In vitro experiments have shown
preservation (survivin-DEx3) or a marked reduction (survivin-2B) of that survivin binds and inhibits the cell death effector proteases
the antiapoptotic effects known from the “regularly” spliced survivin caspase-3 and caspase-7 (5), which induce a wide range of cellular
isoform. degradation processes determining the morphological phenotype of
apoptosis. Moreover, survivin has been demonstrated to associate
Discussion
specifically with microtubules of the mitotic spindle (15). Because
In this study, we show that human RCC cell lines express the caspase-3 is involved in proteolysis of mitotic spindle proteins (16),
apoptosis inhibitor survivin and extend a recent report by Tamm et al. survivin might assist to preserve the integrity of the mitotic apparatus,
(5) on survivin expression in RCCs of the clear cell type to human thereby cooperating with other components of the G2-M checkpoint.
RCCs of all major histological types. More importantly, however, we Overexpression of survivin in cancer might negatively interfere with
identified for the first time two novel survivin splice variants, which the G2-M checkpoint and promote the progression of neoplastic cells
exhibited either largely preserved (survivin-DEx3) or markedly re- through mitosis (15).
duced (survivin-2B) antiapoptotic properties in transfection experi- Although at least some of its antiapoptotic modes of action are com-
ments. These previously unknown splice variants could not be de- mon to other IAP family members, survivin additionally exhibits struc-
tected by Northern blot hybridization, because only 118-bp (survivin- turally unique features that also have important functional implications.
DEx3) or 69-bp (survivin-2B) differences in length were found when Thus, survivin lacks a COOH-terminal RING finger, which appears to be
compared with the primarily identified survivin transcript. critical for the antiapoptotic function of baculoviral and some cellular
Recently, survivin has attracted considerable attention as a novel IAPs (2). Moreover, survivin contains a single BIR domain only, in
member of the IAP family. At variance with the ubiquitous distribution of contrast to other human IAP family members identified thus far (2). In
other IAPs in fetal and adult tissues, survivin expression was shown to be this context, it was striking to note that the novel splice variants survivin-
developmentally regulated, with intensive expression in fetal tissues and DEx3 and survivin-2B exhibited pronounced structural alterations of the
complete down-regulation in most adult tissues (7). Most strikingly, single BIR domain. Deletion of exon 3 in survivin-DEx3 results in a
however, reexpression of survivin was found in the most common human frame shift with truncation of the BIR domain in addition to a new
6100
 NOVEL SPLICE VARIANTS OF SURVIVIN

Fig. 3. Differential alterations of protein domains in the novel survivin splice variants. a, protein sequences were analyzed with the Prosite program of the PC/Gene package (21).
The BIR domain, which is known to be responsible for inhibition of certain caspases, was found to be modified in both alternative splice variants of survivin by computer analysis.
Survivin-2B has acquired additional 23 amino acids encoded by the cryptic exon 2B. The novel domain introduces a potential N-glycosylation site as well as two potential
N-myristoylation sites. Skipping of exon 3 in survivin-DEx3 results in a frame shift that causes a new COOH-terminal sequence with a potential N-myristoylation site. BIR9, modified
BIR; PKC, protein kinase C phosphorylation site; N-Myr, N-myristoylation site; N-Glyc, N-glycosylation site; CK2, casein kinase 2 phosphorylation site; cAMP, cyclic AMP-dependent
kinase phosphorylation site. b, multiple alignment, using Clustal W (22) and enhanced with Boxshade, of amino acid sequences of the three survivin variants. Darker shading is of
residues that are highly conserved; lighter shading is of less well-conserved residues, and residues that are not shaded are not conserved.

COOH-terminal protein segment, which in turn might affect functional receptor family exist in membrane-bound and soluble isoforms, which
properties such as subcellular localization (compare Fig. 3). Despite these antagonistically affect apoptosis. The Bcl-2-family encompasses genes,
structural modifications, survivin-DEx3 exhibited antiapoptotic proper- e.g., bcl-x, the different splice variants of which can antagonistically
ties similar to those of survivin in our experimental system. However, determine the susceptibility for cell death signals (18). Finally, different
survivin-DEx3-mediated antiapoptosis in HepG2 cells might be a cell isoforms of caspases, the executive proteins of apoptosis, are generated
type-specific phenomenon. Nevertheless, the BIR domain alone was by alternative splicing (19).
observed to be sufficient for inhibition of apoptosis in many other Although alternative splicing of survivin considerably adds to the
systems using the cellular IAPs (2). Therefore, we cannot conclusively complexity of the systems controlling apoptosis, there might be an
explain, thus far, why survivin-DEx3 still exerted antiapoptotic effects additional level of regulation. Thus, the survivin gene had been
with a single truncated BIR domain. This observation, however, might be identified by hybridization with the cDNA encoding the EPR-1, and
related either to preserved antiapoptotic potential of the BIR residues or the coding sequence of survivin was found to be largely complemen-
to as yet unknown antiapoptotic properties in other regions of the sur- tary to that of EPR-1. This observation suggested the possibility of
vivin-DEx3 molecule. apoptosis regulation by naturally occurring antisense interactions be-
In contrast, the acquisition of an additional “cryptic” exon in tween survivin and EPR-1 transcripts (20). Although it still remains to
survivin-2B resulted in a modification of the BIR domain (compare be proved that these two transcripts actually interact in vivo, first
Fig. 3), which proved to be functionally relevant in transfection evidence for such a mechanism has come from transfection experi-
experiments. Thus, the potential of survivin-2B to inhibit methotrex- ments in HeLa cells (20). In these experiments, induction of EPR-1
ate-induced apoptosis in HepG2 cells was markedly reduced, possibly mRNA suppressed the expression of endogenous survivin, which was
because of a dominant-negative mechanism of competitive binding to followed by increased apoptosis and reduction of cell number. Further
the interaction partners of survivin. investigations will have to show whether the newly identified splice
Although further experimental work will have to elucidate the func- variants might additionally modify these postulated antisense interac-
tional properties of the different survivin splice variants in more detail, tions in vitro and in vivo.
our observations indicated for the first time that alternative splicing might Taken together, the identification of two novel splice variants will
be relevant for the fine tuning of survivin actions and possibly for the have profound implications for our understanding of survivin actions.
actions of other IAPs as well. Alternative splicing has been found pre- These splice variants may not only be involved in the regulation of
viously to play a key role in the regulation of apoptosis, determining the apoptosis during fetal development but may also determine the re-
actions of many apoptosis-related genes along all levels of the apoptotic sponse of cancer cells to apoptosis-inducing stimuli such as anticancer
pathway (reviewed in Ref. 17). Thus, members of the CD95 death drugs and irradiation. Elucidation of the mechanisms controlling the
6101
 NOVEL SPLICE VARIANTS OF SURVIVIN

expression and alternative splicing of survivin, therefore, could facil-

itate the disruption of a potent antiapoptotic mechanism in cancer
cells. This intervention could selectively increase the susceptibility of
cancer cells to apoptosis-based treatment strategies without affecting
viability of nonneoplastic tissues that do not express survivin.

Acknowledgments

We thank Dr. Dario Altieri for providing the antibody to human survivin
and Siegrid Khalil, Martina Bellack, and Michael Ringler for excellent tech-
nical assistance. The results are part of the Ph.D. thesis of Michael Wenzel.

References
1. Rudin, C. M., and Thompson, C. B. Apoptosis and disease: regulation and clinical
relevance of programmed cell death. Annu. Rev. Med., 48: 267–281, 1997.
2. LaCasse, E. C., Baird, S., Korneluk, R. G., and MacKenzie, A. E. The inhibitors of
apoptosis (IAPs) and their emerging role in cancer. Oncogene, 17: 3247–3259, 1998.
3. Rothe, M., Pan, M. G., Henzel, W. J., Ayres, T. M., and Goeddel, D. V. The
TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral
inhibitor of apoptosis proteins. Cell, 83: 1243–1252, 1995.
4. Ambrosini, G., Adida, C., and Altieri, D. C. A novel anti-apoptosis gene, survivin,
expressed in cancer and lymphoma. Nat. Med., 3: 917–921, 1997.
5. Tamm, I., Wang, Y., Sausville, E., Scudiero, D. A., Vigna, N., Oltersdorf, T., and
Reed, J. C. IAP-family protein survivin inhibits caspase activity and apoptosis
induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res., 58:
5315–5320, 1998.
6. Cohen, G. M. Caspases: the executioners of apoptosis. Biochem. J., 326: 1–16, 1997.
7. Adida, C., Crotty, P. L., McGrath, J., Berrebi, D., Diebold, J., and Altieri, D. C.
Developmentally regulated expression of the novel cancer anti-apoptosis gene sur-
vivin in human and mouse differentiation. Am. J. Pathol., 152: 43– 49, 1998.
8. Adida, C., Berrebi, D., Peuchmaur, M., Reyes-Mugica, M., and Altieri, D. C.
Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet, 351: 882–
883, 1998.
9. Kawasaki, H., Altieri, D. C., Lu, C. D., Toyoda, M., Tenjo, T., and Tanigawa, N.
Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer.
Cancer Res., 58: 5071–5074, 1998.
10. Gerharz, C. D., Moll, R., Störkel, S., Ramp, U., Thoenes, W., and Gabbert, H. E.
Ultrastructural appearance and cytoskeletal architecture of the clear cell, chro-
mophilic and chromophobe cell variants of human renal cell carcinomas in vivo and
in vitro. Am. J. Pathol., 142: 851– 859, 1993.
11. Ramp, U., Jaquet, K., Reinecke, P., Nitsch, T., Gabbert, H. E., and Gerharz, C. D.
Acquisition of TGF-b1 resistance: an important progression factor in human renal cell
carcinoma. Lab. Investig., 76: 739 –749, 1997.
12. Müller, M., Strand, S., Hug, H., Heinemann, E. M., Walczak, H., Hofmann, W. J.,
Stremmel, W., Krammer, P. H., and Galle, P. R. Drug-induced apoptosis in hepatoma
cells is mediated by the CD95 (APO- 1/Fas) receptor/ligand system and involves
activation of wild-type p53. J. Clin. Investig., 99: 403– 413, 1997.
13. Staden, R. Computer methods to locate signals in nucleic acid sequences. Nucleic
Acids Res., 12: 505–519, 1984.
14. Mount, S. M. A catalogue of splice junction sequences. Nucleic Acids Res., 10:
459 – 472, 1982.
15. Li, F., Ambrosini, G., Chu, E. Y., Plescia, J., Tognin, S., Marchisio, P. C., and Altieri,
D. C. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature
(Lond.), 396: 580 –584, 1998.
Fig. 4. Differential antiapoptotic potential of the novel survivin splice variants. a, 16. Andrade, F., Roy, S., Nicholson, D., Thornberry, N., Rosen, A., and Casciola-Rosen,
coding sequences of survivin and its two splice variants were ligated into the L. Granzyme B directly and efficiently cleaves several downstream caspase sub-
expression vector pEGFP-N3 and transfected into the human hepatoma cell line strates: implications for CTL-induced apoptosis. Immunity, 8: 451– 460, 1998.
HepG2. Cell death was induced in transient transfectants by exposure to methotrexate 17. Jiang, Z., and Wu, J. Y. Alternative splicing and programmed cell death. Proc. Soc.
(100 mg/ml). Survivin-DEx3 transfectants exhibited cell survival frequencies closely Exp. Biol. Med., 220: 64 –72, 1998.
corresponding to that of survivin transfectants. In contrast, survivin-2B transfectants 18. Boise, L. H., Gonzalez-Garcia, M., Postema, C. E., Ding, L., Lindsten, T., Turka,
showed a marked reduction of cell survival. The transfection efficiency was $50% in L. A., Mao, X., Nunez, G., and Thompson, C. B. Bcl-x, a bcl-2-related gene that
all transfection experiments. The percentage of transfected cells was determined by functions as a dominant regulator of apoptotic cell death. Cell, 74: 597– 608, 1993.
microscopic evaluation of b-galactosidase expressing cells. Data are the means of at 19. Srinivasula, S. M., Ahmad, M., Guo, Y., Zhan, Y., Lazebnik, Y., Fernandes-Alnemri,
least three independent experiments; bars, SD. Statistical analysis was performed with T., and Alnemri, E. S. Identification of an endogenous dominant-negative short
Dunnett’s test. Of note, corresponding results were obtained using untagged isoform of caspase-9 that can regulate apoptosis. Cancer Res., 59: 999 –1002, 1999.
pcDNA3.1-survivin/survivin-DEx3/survivin-2B constructs (data not shown). b, im- 20. Ambrosini, G., Adida, C., Sirugo, G., and Altieri, D. C. Induction of apoptosis and
munoblot analysis with both anti-GFP and anti-survivin antibodies revealed stable and inhibition of cell proliferation by survivin gene targeting. J. Biol. Chem., 273:
comparable levels of expression for the indicated survivin variants in HepG2 trans- 11177–11182, 1998.
fectants. Of note, binding of anti-GFP antibody is independent of the survivin 21. Bairoch, A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids
component of the fusion protein. Additional survivin-2B bands detected by the Res., 19: 2241–2245, 1991.
anti-GFP antibody might indicate posttranslational modifications of this survivin 22. Thompson, J. D., Higgins, D. G., and Gibson, T. J. CLUSTAL W: improving the
variant. The polyclonal anti-survivin antibody, which was generated against “regular” sensitivity of progressive multiple sequence alignment through sequence weighting,
survivin, detected survivin splice variants with less intensities, as evident from positions-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22:
comparison with a-tubulin controls. 4673– 4680, 1994.

6102