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Blood, 1 December 2000, Vol. 96, No. 12, pp. 4002-4003
CORRESPONDENCE
To the editor:
Survivin does not inhibit caspase-3 activity
Conway et al recently reported that alternatively spliced forms
of mouse survivin exhibit different antiapoptotic
properties.1 This was inferred from inhibition of
recombinant caspase-3 catalytic activity using a standard chromogenic
assay in the presence of increasing concentrations of
mammalian-expressed murine survivin isoforms of 140, 121, and 40 amino
acids, respectively.1 If confirmed, the ability of
survivin to inhibit caspase-3 activity would have a major impact on
targeting this ubiquitous cytoprotection pathway in
cancer2,3 and during cell cycle progression.4 But the data of survivin inhibition of caspase-3 activity presented by
Conway et al raise serious concerns of specificity.1
First, the experiments contained no controls with genuine inhibitors of
caspase-3, including DEVD-CHO or another IAP family protein with
well-documented anti-caspase-3 activity (ie, XIAP). Second, from the
data presented it was impossible to derive an inhibition constant
(Ki) of the cleavage reaction, which is indispensable to
quantitatively characterize potential caspase inhibitors.5 We have now reinvestigated the data presented by Conway et
al1 and attempted to reproduce their caspase-3 inhibition
experiments using mouse or human survivin proteins. Mouse recombinant
survivin was expressed, purified to homogeneity (Figure
1A), and properly folded by 1D-NMR
analysis. Concentrations of recombinant mouse survivin up to 80 µmol/L failed to decrease purified, recombinant caspase-3 activity
using a peptide substrate cleavage assay similar to that reported by
Conway et al1 (Figure 1B). In contrast, 0.1 µmol/L XIAP
completely inhibited caspase-3 activity, in agreement with previous
data.5 In an attempt to reproduce exactly the mammalian
cell expression approach used by Conway et al1, we immunoaffinity-purified native survivin from Jurkat T cells. Eluted fractions contained a single 16.5-kd survivin band by immunoblotting with an antibody to survivin (Figure 1C). But increasing concentrations of native, immunoaffinity-purified survivin did not affect caspase-3 catalytic activity, as determined by substrate peptide cleavage (Figure 1D).
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| Figure 1.
Survivin does not inhibit caspase-3 activity.
(A) SDS gel electrophoresis of purified mouse recombinant
survivin. (B) Effect of murine survivin or human XIAP on caspase-3
activity by hydrolysis of the fluorogenic substrate, DEVD-AMC. (C)
Western blotting of immunoaffinity-purified human survivin isolated
from Jurkat T cells by chromatography on  -survivin-sepharose. (D)
Effect of native human survivin on caspase-3 activity by hydrolysis of
the fluorogenic substrate, DEVD-AMC. W, whole cell extracts.
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In conclusion, our data strongly argue against any role of survivin in
directly inhibiting caspase-3 activity, at variance with the
preliminary work of Conway et al.1 These discrepancies cannot be ascribed to species specificity, protein purification, or
differences in experimental protocol. Moreover, available structural data demonstrate that a linker region upstream of the second
baculovirus IAP repeat is required for docking IAP proteins (ie, XIAP)
to active caspase-3.6 This linker region is absent in
survivin, thus further weakening the hypothesis proposed by Conway et
al.1 Therefore, the general conclusion of Conway et
al1 that alternatively spliced isoforms of survivin have
different antiapoptotic functions is not experimentally substantiated.
The means by which survivin participates in the apoptosis balance in
cancer and during cell cycle progression require further investigation.
Damon P. Banks, Janet Plescia, and Dario C. Altieri
Department of Pathology Boyer Center for Molecular
Medicine Yale University School of Medicine New Haven, CT
Jun Chen, Saul H. Rosenberg, Haichao Zhang, and Shi-Chung Ng
Cancer Research Abbott Laboratories Abbott Park, IL
Acknowledgments
Supported in part by National Institutes of Health grant CA78810.
References
1.
Conway EM, Pollefeyt S, Cornelissen J, et al.
Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions.
Blood.
2000;95:1435-1442[Abstract/Free Full Text].
2.
Ambrosini G, Adida C, Altieri DC.
A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.
Nat Med.
1997;3:917-921[Medline]
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3.
Velculescu VE, Madden SL, Zhang L, et al.
Analysis of human transcriptomes.
Nat Genet.
1999;23:387-388[Medline]
[Order article via Infotrieve].
4.
Li F, Ambrosini G, Chu EY, et al.
Control of apoptosis and mitotic spindle checkpoint by survivin.
Nature.
1998;396:580-584[Medline]
[Order article via Infotrieve].
5.
Deveraux QL, Reed JC.
IAP family proteins-suppressors of apoptosis.
Genes Dev.
1999;13:239-252[Free Full Text].
6.
Sun C, Cai M, Gunasekera AH, et al.
NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP.
Nature.
1999;401:818-822[Medline]
[Order article via Infotrieve].
Response:
Evidence that survivin inhibits caspase-3 activity
Earlier this year, we provided a definitive report indicating
that there exist at least 3 murine survivin mRNA variants, each encoding a distinct protein.1 We demonstrated, with
appropriate controls, that both survivin140 and
survivin121 are able to inhibit caspase-3 activity, while
survivin40 does not. In contrast to the claims of Altieri's group in their letter, several
reports support our finding that survivin interferes with caspase-3
activity. These include the following: Reed's group reported that
"survivin was able to substantially reduce caspase activity, as
measured by cleavage of a tetrapeptide substrate, AspGluValAsp-aminofluorocoumarin. Similar results were obtained in
intact cells when Survivin was overexpressed."2(p5315)
Altieri's group noted in their Nature paper that
"like other IAP proteins, survivin inhibits the terminal effectors
caspase-3 and caspase-7."3(p583) Kobayashi et al
demonstrated that overexpression of murine survivin in Rat1 cells
inhibited caspase-induced cell death and also that a purified GST
fusion protein encoding murine survivin could bind directly to
caspase-3.4 This group further transfected Jurkat cells
with epitope-tagged survivin and showed by immunoprecipitation with
anti-caspase-3 antibodies that survivin "can bind efficiently to
processed caspase 3."4(p1460) In their
Nature Cell Biology paper, Altieri's group once again noted
that survivin regulates apoptosis via caspase-3: "Expression of
survivin (C84A) or survivin antisense cDNA also resulted in increased
activity of the apoptosis effector caspase-3, as judged by hydrolysis
of the fluorogenic caspase-3 substrate."5(p461)
Altieri's group also performed studies on cultured endothelial cells and reported that "[r]ecombinant expression of green
fluorescent protein survivin in endothelial cells reduced caspase-3
activity and counteracted apoptosis induced by tumor necrosis
factor."6(p393) And finally, a recent report
confirms that the down-regulation of survivin mRNA levels by an
antisense approach results in increased caspase-3
activity.7 We note that many of the data indicating that survivin interferes with
caspase-3 activity predate our own and, in several cases, have actually
been provided by Altieri's group. We are not in a position to assess
fully the experiments described by Altieri's group and would point out
only that they are not identical to our own, in contrast to what that
group claims. In addition, we would add that we have observed that the
activity of recombinant survivin is highly dependent on the method
chosen for its synthesis and purification. Based on our experiments and
the current published data, we believe that specific forms of survivin
do inhibit caspase-3 activity. Nevertheless, we agree that the means by
which survivin participates in the apoptosis balance requires
further investigation, and we hypothesize that the alternatively
spliced isoforms of survivin may modulate apoptosis differentially.
Edward M. Conway and Désiré Collen
Center for Transgene Technology and Gene Therapy
Flanders Interuniversity Institute for Biotechnology
University of Leuven, Leuven, Belgium
Andre Schuh
Department of Medicine and the Institute of Medical Science
University of Toronto Toronto, Ontario, Canada
References
1.
Conway EM, Pollefeyt S, Cornelissen J, et al.
Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions.
Blood.
2000;95:1435-1442.
2.
Tamm I, Wang Y, Sausville E, et al.
IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs.
Cancer Res.
1998;58:5315-5320[Abstract/Free Full Text].
3.
Li F, Ambrosini G, Chu EY, et al.
Control of apoptosis and mitotic spindle checkpoint by survivin.
Nature.
1998;396:580-584.
4.
Kobayashi K, Hatano M, Otaki M, Ogasawara T, Tokuhisa T.
Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation.
Proc Natl Acad Sci U S A.
1999;96:1457-1462[Abstract/Free Full Text].
5.
Li F, Ackermann EJ, Bennett CF, et al.
Pleiotropic cell-division defects and apoptosis induced by interference with survivin function.
Nat Cell Biol.
1999;1:461-466[Medline]
[Order article via Infotrieve].
6.
O'Connor DS, Schechner JS, Adida C, et al.
Control of apoptosis during angiogenesis by survivin expression in endothelial cells.
Am J Pathol.
2000;156:393-398[Abstract/Free Full Text].
7.
Olie RA, Simoes-Wust AP, Baumann B, et al.
A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy.
Cancer Res.
2000;60:2805-2809[Abstract/Free Full Text].
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