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 mmol/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 mmol/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). 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.