DIABLO in ovarian carcinoma cells but is redundant in Smac-mediated apoptosis

Experimental Cell Research 302 (2005) 69 – 82 www.elsevier.com/locate/yexcr

Survivin interacts with Smac/DIABLO in ovarian carcinoma cells but is redundant in Smac-mediated apoptosis I.A. McNeisha,*, R. Lopesa, S.J. Bella, T.R. McKaya,1, M. Fernandeza, M. Lockleya, S.P. Wheatleyb, N.R. Lemoinea a

Cancer Research UK Molecular Oncology Unit, Barts and the London School of Medicine, London EC1M 6BQ, United Kingdom b Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RH, UK Received 2 June 2004, revised version received 19 August 2004 Available online 18 September 2004

Abstract Abnormalities in the control and execution of apoptosis are seen in many malignancies, including ovarian carcinoma. Many of these abnormalities involve the mitochondrial pathway of apoptosis, including overexpression of BIR-containing inhibitor of apoptosis protein (IAP) family proteins as well as dysregulated apoptosome function. We sought to stimulate the mitochondrial pathway of apoptosis by constructing a recombinant adenovirus encoding mature, processed Smac/DIABLO (Ad CMV tSmac), the second mitochondrial activator of caspases. Transfection of ovarian carcinoma cells with Ad CMV tSmac leads to increasing apoptosis in a dose-dependent manner. By contrast, transfection of IOSE397 immortalized normal ovarian surface epithelial cells does not cause apoptosis. We also show that the processed form of Smac is primarily expressed in the cytosol of ovarian carcinoma cells. Smac co-immunoprecipitates with both survivin and XIAP and stimulates survivin, but not XIAP, down-regulation. This down-regulation does not result from transcriptional changes, as determined by quantitative real-time PCR, but cycloheximide treatment indicates that survivin half-life is reduced from 6 to 2 h, which is secondary to ubiquitination and proteasomal degradation. RNA interference, however, suggests that survivin does not act to inhibit Smacmediated apoptosis, which is confirmed by cotransfection with the phosphorylation mutant, survivin T34A. Finally, intraperitoneal delivery of Ad CMV tSmac increases median survival of mice bearing human ovarian carcinoma xenografts. We believe that expression of Smac/ DIABLO can stimulate the intrinsic pathway of apoptosis in ovarian carcinoma without damaging normal ovarian tissue and therefore has therapeutic potential. D 2004 Elsevier Inc. All rights reserved. Keywords: Survivin; Smac; Ovarian carcinoma cells

Introduction Dysregulated apoptosis is observed in many malignancies [1], including ovarian cancer, where multiple abnormalities have been reported, including overexpression of members of the inhibitor of apoptosis protein (IAP) family

* Corresponding author. Cancer Research UK Molecular Oncology Unit, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK. Fax: +44 20 7014 0431. E-mail address: [email protected] (I.A. McNeish). 1 Present address: Cystic Fibrosis/Pulmonary Research and Treatment Center, University of NC, Chapel Hill, NC, USA. 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.08.029

and abnormal apoptosome function [2–4]. Abnormal apoptosis is especially evident in drug-resistant cells [5,6]. The IAP family of proteins acts to down-regulate apoptosis and all true IAPs contain at least one BIR (baculovirus IAP repeat) domain, which is crucial to their antiapoptotic function. In XIAP, the linker between BIR1 and BIR2 can inhibit activated caspase-3 [7], while BIR3 is capable of inhibiting caspase-9 [8]. IAP activity is, in turn, controlled in several ways. The proteins are subjected to proteasomal degradation and have intrinsic E3 ubiquitin ligase activity mediated by a RING motif and can autoubiquitinate [9]. Another level of control is mediated by proteins bearing an N-terminal tetrapeptide motif called RHG after the three

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Drosophila genes, reaper, hid, and grim. The products of these three genes are required for apoptosis induction in Drosophila and function by binding to and inhibiting the BIR domains of IAP proteins. Smac/DIABLO was the first mammalian RHG protein to be described [10,11], and many studies have since demonstrated it too can inhibit IAPs by direct binding. After translation, full-length Smac translocates to the intermembrane space of mitochondria, where the N-terminal 55 amino acids that constitute the mitochondrial localization signal are removed to reveal the RHG motif, AVPI [10,11]. Smac is then released from mitochondria in response to many apoptotic stimuli, both those that activate the death receptor [12] and intrinsic pathways [13]. However, its precise physiological role in mammalian apoptosis remains unclear. Smac-deficient mice have an essentially normal phenotype, which strongly suggests that other mammalian proteins can perform its IAP-inhibiting function [14]. Omi/HtrA2 [15,16] and more recently proteolytically cleaved GDPT1/eRF3 [17], are obvious candidates for this molecular redundancy, and caspase activity can cleave other cellular proteins to reveal RHGtype motifs [18]. Nonetheless, Smac may have potential as a novel therapeutic in cancer. It is capable of potentiating the proapoptotic activity of epothilone B, [19], TRAIL [20], and a wide range of chemotherapeutic drugs [21], although some previous reports have suggested that it cannot induce apoptosis alone, even when the mature, processed form is expressed within the cytosol [10,22]. Survivin is unquestionably a controversial protein. Some reports have suggested that it is an IAP with a purely antiapoptotic role due to the presence of a BIR domain [23], that it is capable of binding directly to caspases 3 and 7 [24], and that disruption of its activity via phosphorylation mutants [25], antisense oligonucleotides [26], or ribozymes [27] can induce apoptosis both in vitro and in vivo. Recent evidence suggests that its main role is actually that of mitotic spindle regulator [28–30] via interactions with other chromosomal passenger proteins such as INCENP and Aurora B kinase [31–33]. However, the possibility that survivin may still have a function in apoptosis has not disappeared; for example, it was recently shown that it was capable of binding to and inhibiting caspase-9 indirectly, with the hepatitis B X protein-interacting protein (HBXIP) acting as intermediary [34] and that it could induce apoptosis by causing nuclear translocation of apoptosisinducing factor (AIF) [35]. It is postulated that survivin may therefore be bifunctional, acting to suppress apoptosis if located in the cytoplasm and acting as a chromosomal passenger protein in G2/M [36]. We have previously demonstrated that an adenovirus encoding full-length Smac is capable of inducing apoptosis in ovarian carcinoma cells [37]. This apoptosis did not require the release of cytochrome c from mitochondria and was not inhibited by Bcl-2 overexpression. We have sought to extend these data. We have generated a recombinant adenovirus encoding the mature, processed form of Smac as

well as a Smac mutant (GVPI-Smac) in which the first alanine of the RHG motif is substituted with glycine to prevent IAP binding. We show that Smac, but not GVPISmac, is a potent inducer of apoptosis in ovarian carcinoma cells but not normal ovarian epithelial cells and that Smac interacts with survivin in ovarian carcinoma cells. This leads to survivin ubiquitination and proteasomal degradation. However, results suggest that survivin does not play an inhibitory role in Smac-mediated apoptosis. Finally, we show that adenoviral-delivered Smac has therapeutic activity in vivo.

Results Expression of Smac induces apoptosis in ovarian carcinoma cells but not normal ovarian epithelial cells We postulated that transfection of ovarian carcinoma cells with an adenovirus encoding processed Smac (Ad CMV tSmac), lacking the 55 N-terminal amino acid mitochondrial localization signal, could induce apoptosis because Smac would not require processing and would locate primarily in the cytoplasm. Therefore, ovarian carcinoma cells were transfected with Ad CMV tSmac at multiplicities of infection (MOI) 10–100 pfu/cell and cell viability was assessed 72 h later by MTT assay (Fig. 1A). Ad CMV tSmac produced a dose-related reduction in cell viability and FACS analysis indicated the presence of a subG1 population suggestive of apoptosis (Fig. 1B). Similar reductions in survival were also observed when OVCAR3, A2780, and A2780CP ovarian carcinoma cells were transfected with Ad CMV tSmac (data not shown). Transfection with a virus encoding a Smac mutant that contains an alanine–glycine substitution at the first amino acid of the RHG motif causes some reduction in cell survival and apoptosis, but this is significantly less than is caused by the same MOI of Ad CMV tSmac (Figs. 1A and B). To examine whether Smac induced cell death or apoptosis in nonmalignant cells, we also transfected IOSE397 immortalized ovarian surface epithelial cells with both Ad CMV tSmac and Ad CMV GVPI-tSmac at MOI 10–100. MTT assays indicate that there is minimal cytotoxicity (Fig. 1A), while cell cycle analysis shows no apoptosis induction (data not shown). FACS analysis of IOSE397 cells following transfection with Ad CMV GFP indicates that they are at least as infectable as IGROV1 or OVCAR4 cells with Ad5-based vectors (data also not shown). Western blot analysis of IGROV1 cells treated with Ad CMV tSmac for 48 h shows cleavage of caspases-9 and -3, with cleavage of PARP, suggesting that the activated caspase-3 is functional (Fig. 1C). At the higher MOI of Ad CMV tSmac, there is also some demonstrable caspase-8 cleavage, although this may be secondary to prior caspase-3 activation (see Discussion).

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Fig. 1. Adenoviruses encoding Smac/DIABLO induce apoptosis in ovarian carcinoma cells. (A) OVCAR4, IGROV1, and IOSE397 cells were transfected with Ad LM-X MOI 100 (X), Ad CMV GVPI-tSmac, Ad CMV tSmac (both MOI 10, 30, and 100), or mock-infected (M). Cell survival was assessed 72 h later by MTT assay. Results represent mean F SD, n = 4. (B) IGROV1 cells were transfected with Ad CMV GFP, Ad LM-X, Ad CMV GVPI-tSmac, or Ad CMV tSmac (all MOI 30) or mock infected. They were harvested 72 h posttransfection and analyzed for cell-cycle status following propidium iodide staining. Numbers represent the percentage of whole cell population in sub-G1. (C) The 106 IGROV1 cells were transfected with either Ad LM-X (MOI 30) or Ad CMV tSmac (MOI 10 and 30). Thirty micrograms protein was separated on SDS-PAGE gels and analyzed by immunoblot.

We next investigated the localization of Smac in OVCAR4 cells following transfection with Ad CMV tSmac, Ad CMV GVPI-tSmac, and the control adenovirus Ad LM-X using cytosolic and mitochondrial subcellular

fractions. Whilst the expression of Smac in the mitochondria was similar following all three transfections, significant amounts of processed 21 kDa Smac were detectable in the cytosol only after transfection with the two Smac viruses

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Fig. 2. Processed Smac is expressed in the cytosol. OVCAR4 cells were transfected with Ad LM-X, Ad CMV tSmac, and Ad CMV GVPI-tSmac (all MOI 20). Forty-eight hours thereafter, cells were harvested and cytosolic and mitochondria-enriched cell fractions isolated. Ten micrograms of each fraction was separated on 12% SDS-PAGE gels and analyzed by immunoblot.

(Fig. 2). Transfection with both Smac viruses also led to a minor release of cytochrome c into the cytosol (see Discussion). Smac/DIABLO interacts with both XIAP and survivin and causes posttranslational down-regulation of survivin Following transfection with Ad CMV tSmac at MOI 30, Western blotting demonstrated marked reduction in the expression of survivin (Fig. 3A), but not the other BIRcontaining proteins XIAP, cIAP-1, or c-IAP-2. To investigate this further, we first performed quantitative real-time PCR for Smac, XIAP, c-IAP1, and survivin in IGROV1 cells transfected with Ad CMV tSmac or the control virus Ad LM-X (Fig. 3B). As expected, there is an increase in Smac mRNA of up to 1000-fold following transfection with Ad CMV tSmac, while transfection with a control adenovirus causes a small, but significant, increase in expression of all the genes studied. However, Ad CMV tSmac transfection did not cause any significant change in the expression of XIAP, c-IAP1, or survivin, implying that reduced transcription cannot explain the down-regulation of survivin seen on Western blot. Therefore, to explore possible posttranscriptional events, we first looked to see whether Smac associated with BIRcontaining proteins in ovarian carcinoma cells. Immunoprecipitation for Smac was performed in IGROV1 cells transfected with both Ad CMV tSmac and Ad CMV GVPI-tSmac, followed by immunoblotting for survivin and XIAP. We were able to demonstrate that Smac, but not GVPI-Smac, coimmunoprecipitated with both XIAP and survivin (Fig. 3C). It was not possible to retrieve native survivin by immunoprecipitation so we utilized a Tap-tagged construct, pcDNA3Tap-Survivin. IGROV1 cells were transfected with both Ad CMV tSmac and pcDNA3-Tap-Survivin and were lysed 48 h later and subjected to tandem affinity purification [38] then immunoblotted for Smac and survivin. These results confirm that Smac is capable of interacting with survivin as the immunoprecipitation had suggested (Fig. 3D). We next looked at the impact of Smac on survivin stability as we have previously shown that the ubiquitin-proteasome pathway is fundamental to the control of survivin degradation [39]. IGROV1 cells were transfected with Ad CMV tSmac in the presence and absence of 50 Ag/ml cycloheximide and cell

lysates were analyzed for expression of survivin and XIAP as well as another chromosomal passenger protein, aurora B kinase (Fig. 4A). Following transfection with the control virus, Ad LM-X, survivin half-life is approximately 6 h (Fig. 4B). However, in the presence of Smac, survivin half-life is significantly reduced to approximately 2 h and this can be reversed in the presence of the 20-S proteasome inhibitor lactacystin at a concentration of 12.5 AM. This strongly implies that survivin is being targeted for proteasomal degradation in the presence of Smac. By contrast, the halflife of both XIAP and aurora B kinase did not reduce significantly following transfection with Ad CMV tSmac (Fig. 4C). Interestingly, when cells were transfected with Ad CMV GVPI-tSmac, there was a reduction in survivin half-life from 6 h to approximately 3.5 h, although this difference did not reach statistical significance (see Supplementary figure). To explore the possibility that survivin degradation via ubiquitination is promoted when Smac is expressed, we performed in vitro and in vivo ubiquitination assays (Fig. 5). When Tap-survivin is added alone to the in vitro reaction, there is a modest amount of polyubiquitination, as demonstrated by the characteristic smear on immunoblot, which suggests that either there is some autoubiquitination of survivin or other proteins bound to survivin can mediate this effect. Nonetheless, the ubiquitination increases greatly when both Smac and purified Tap-survivin are present in the reaction, reinforcing the suggestion that Smac promotes the ubiquitination of survivin. The in vitro reaction was repeated using either Smac immunoprecipitate or an equivalent amount of recombinant His-tagged human Smac. It was noted that the polyubiquitin smear was far greater in the presence of the Smac immunoprecipitate, suggesting that other Smac-binding proteins may contribute to the survivin ubiquitination. To explore events in vivo, OVCAR 4 cells were transfected with Ad CMV tSmac in the presence of survivin and ubiquitin in the presence or absence lactacystin, which was added 16 h before harvest. When the proteasome inhibitor was present, there was a marked increase in the smear of ubiquitinated survivin seen on immunoblot. Survivin does not inhibit Smac-mediated apoptosis Next, we sought to investigate whether the binding of survivin to Smac inhibited Smac-mediated cytotoxicity using RNA interference, employing siRNA oligonucleotides. Exposure of IGROV1 cells to 30–120 pmol of survivin siRNA oligonucleotides for 72 h led to a marked reduction in both survivin mRNA and protein (Fig. 6A) compared to untransfected cells or those transfected with control siRNA. Of note, the survivin siRNA has no demonstrable effect on XIAP expression. However, following transfection with 30 and 60 pmol survivin siRNA, Ad CMV tSmac-mediated toxicity was not significantly altered (Fig. 6B) compared to mock transfected or control siRNAtransfected cells. It was noted that 60 pmol survivin RNAi

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Fig. 3. Smac causes survivin down-regulation and co-immunoprecipitates with both XIAP and survivin. (A) Forty-eight hours after transfection with Ad LM-X MOI 30 (X) or Ad CMV tSmac MOI 10 and 30, IGROV1 cells were harvested and 30 Ag protein was separated on SDS-PAGE gels and analyzed by immunoblot. (B) Total cellular RNA was harvested from IGROV1 cells 48 h following mock transfection (control) or transfection with Ad LM-X MOI 30 or Ad CMV tSmac MOI 10 and 30. Semiquantitative real-time PCR was performed as outlined in Materials and methods, using 18S ribosomal RNA as internal control. Results are presented relative to mock-transfected cells and represent mean F SD, n = 3. (C) IGROV1 cells were transfected with Ad LM-X (X), Ad CMV GVPI-tSmac (GVPI), or Ad CMV tSmac (tS), all MOI 30. Forty-eight hours later, they were lysed and immunoprecipitated with a rat anti-Smac mAb. The 20 Ag whole cell lysate and 10 Al precipitate were separated on SDS-PAGE gels and analyzed by immunoblot. (D) IGROV1 cells were transfected with Ad LM-X MOI 30 or Ad CMV tSmac MOI 30. Six hours later, they were transfected with either pcDNA3 Tap control (C) or pcDNA3 Tap-survivin (T-S). Fortyeight hours thereafter, cells were lysed and subjected to tandem affinity purification (TAP). Five microliters lysate or 10 Al of TAP-purified protein was separated on 10 or 15% SDS-PAGE gels and analyzed by immunoblot.

did not interfere with the expression of Smac following viral transfection and did not itself cause a reduction in survival of IGROV1 cells compared to control RNAi (Fig. 6B). To explore this further, we cotransfected IGROV1 cells with Ad CMV tSmac and an adenovirus encoding the dominant negative phosphorylation-defective survivin

mutant, survivin T34A. Again, there is no reduction in Smac-mediated toxicity. Finally, we co-transfected IGROV1 with Ad CMV tSmac and a plasmid encoding wild-type survivin and again demonstrated that there was no alteration in Smac-mediated cytotoxicity. In both these latter experiments, the presence of Smac did not hinder the

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Fig. 4. Smac causes reduction in survivin, but not XIAP or Aurora B kinase, half-life. (A) Forty-hours after transfection with Ad LM-X MOI 20 or Ad CMV tSmac MOI 20, IGROV1 cells were washed and refed with medium containing 50 Ag/ml cycloheximide (CHX). They were harvested up to 6 h later and 30 Ag protein was separated on SDS-PAGE gels and analyzed by immunoblot for survivin, XIAP, and Aurora B kinase. Additionally, cells were washed and refed with medium containing 12.5 AM lactacystin 1 h before the addition of cycloheximide. (B and C) Membranes were analyzed by phosphoimager and expression of survivin (B) and XIAP and Aurora B (C) was quantified relative to that of actin.

ectopic expression of either wild-type or mutant survivin (Fig. 6C). Taken together, these results strongly suggest that binding to and inhibiting survivin function is not required for Smac-mediated cytotoxicity in ovarian carcinoma cells.

Smac/DIABLO extends survival in mice bearing human ovarian carcinoma xenografts To investigate the therapeutic potential of Smac in vivo, we injected IGROV1 intraperitoneally into nude female

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significant in comparison with PBS control ( P = 0.009), although the difference with Ad LM-X did not quite reach statistical significance ( P = 0.07). In the daily treatment regime, median survival for PBS-treated mice was 24 days. Again, treatment with the control virus Ad LM-X produced a small but nonsignificant increase in survival to 31.5 days. However, treatment with Ad CMV tSmac produced a significant increase in median survival to 41 days (comparison with control virus: P = 0.02; comparison with PBS treated mice: P = 0.002) (Fig. 7B).

Discussion

Fig. 5. Smac enhances survivin ubiquitination both in vitro and in vivo. (A) Two microliters of Smac immunoprecipitate and/or 5 Al of purified Tapsurvivin was subjected to an in vitro ubiquitination assay. Samples were resolved on a 10% SDS-PAGE gel and immunoblotted for ubiquitin. (B) The in vitro ubiquitination assay was repeated using either 2 Al of Smac immunoprecipitate or 2 Ag of in vitro-translated recombinant His-tagged human Smac. (C) OVCAR4 cells were transfected with either Ad LM-X or Ad CMV tSmac (both MOI 30). Six hours later, they were also transfected with pcDNA-survivin. Twenty-four hours thereafter, cells were refed with medium with or without 12.5 AM lactacystin and harvested 16 h later. Thirty micrograms was separated on an SDS-PAGE gel and analyzed by immunoblot for survivin.

mice followed 72 h later by adenovirus. Two experimental regimes were used. In the first, cells were injected on day 1 followed by three weekly virus injections on days 4, 11, and 18 (dose 5  1010 viral particles per injection). Alternatively, cells were injected on day 1 followed by five daily virus injections on days 4–8 inclusive (dose 1  1010 viral particles per injection). With weekly treatment, median survival in the PBS-treated group was 16 and 18.5 days for those that received the control virus Ad LM-X ( P = NS) (Fig. 7A). Treatment with Ad CMV tSmac almost doubled median survival to 31 days, which was statistically highly

Our results presented here demonstrate unequivocally that the expression of mature, processed Smac alone can be sufficient to induce apoptosis in ovarian carcinoma cells. These data partially contradict those of others [10,20] that Smac alone cannot induce apoptosis in tumor cells. There are three possible explanations. Firstly, this represents a cellspecific phenomenon, whereby ovarian carcinoma cells are particularly sensitive to the effects of Smac; certainly, there was minimal apoptosis in normal ovarian epithelial cells in response to Ad CMV tSmac transfection. It has also been reported that some carcinoma cells overexpress both proand antiapoptotic proteins and that a marginal increase in expression of one proapoptotic compound can tip the overall balance in favor of cell death [40]. Secondly, there may be a dose–response effect. Transfection with adenoviral vectors may simply permit greater transgene expression than simple plasmid transfection. Finally, the use of an adenoviral vector to deliver the Smac gene may be instrumental. It has recently been shown that some adenoviral proteins, especially E4orf4, may be able to induce caspase activation [41]. We have previously found that transfection of some ovarian carcinoma cells (especially those that overexpress procaspase-3) with the control adenovirus Ad LM-X can cause apoptosis and a reduction in cell survival [37]. Therefore, in cells where Smac/DIABLO is present within the cytosol, adenoviral proteins might provide a secondary stimulus and combine with Smac/DIABLO to trigger an apoptotic cascade. Our results also indicated that GVPI-Smac is able to induce some apoptosis, albeit less efficiently than Smac itself. This confirms previous results, which suggested that the RHG motif in Smac is not essential for its proapoptotic effect [42]. It has previously been demonstrated that Smac peptides in which the initial alanine of the RHG motif is mutated to glycine are incapable of binding to either the BIR2 or BIR3 domains of XIAP [43]. The immunoprecipitation results in Fig. 3C support this idea, suggesting that an alternative mechanism is responsible for the toxicity of GVPI-Smac. This will be the subject of future investigation. The apoptosis induced by Smac is marked by cleavage of caspases-9 and -3, which suggests that the intrinsic pathway

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of apoptosis is being activated. Two cleaved forms of caspase-9 exist: the 37-kDa form (cleaved as Asp 315 to reveal the RHG motif ATPF) results from apoptosome activation, and this is further cleaved at Asp 330 by

activated downstream caspases to reveal the 35-kDa form, which is partially resistant to the inhibitory effect of XIAP [44]. In Ad CMV tSmac-transfected cells, only the 35-kDa form is seen, suggesting that caspase-9 is being activated

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Fig. 7. Smac/DIABLO extends survival in mice bearing human ovarian carcinoma xenografts. IGROV1 cells were injected intraperitoneally into nude female mice in groups of eight. Viral injections commenced 72 h later. Mice were treated either once weekly for 3 weeks (dose 5  1010 viral particles per injection; A) or daily for 5 days (dose 1  1010 viral particles per injection; B) and monitored for the accumulation of ascites and general well-being.

secondarily by downstream caspases, possibly due to cytochrome c release seen on cellular subfractionation. However, we believe that this cytochrome c release is not the primary cause of induced apoptosis for three reasons. Firstly, the amount of cytochrome c released is the same following both Ad CMV tSmac and Ad CMV GVPI-tSmac transfection, yet the amount of induced apoptosis is far greater with Ad CMV tSmac. Secondly, we have previously shown clearly that Smac-induced apoptosis was not inhibited by overexpression of Bcl-2 [37]. Thirdly, there is recent evidence that caspase-9 can be activated without being cleaved at Asp 315 [45] and that XIAP inhibits caspase-9 by preventing dimerization [8,46]. Therefore, if Smac can remove XIAP from monomeric caspase-9,

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dimerization and activation of the caspase could occur without the apoptosome-mediated 37-kDa form appearing. At higher MOIs of Ad CMV tSmac transfection, some evidence of caspase-8 cleavage is seen, but we believe this to be secondary to the activity of downstream activated caspase-3 as it is only seen at the higher MOI of Ad CMV tSmac transfection. The possibility of a direct Smac/survivin interaction has been postulated for some time. Smac is capable of binding to the BIR domains of XIAP, c-IAP1, c-IAP2, Melanoma IAP (ML-IAP), and OpIAP from the baculovirus O. pseudotsugata [10,21,47,48], and an interaction with survivin was originally suggested but could not be proven unambiguously [11]. The single BIR domain of survivin bears a close structural homology to BIR2 of XIAP [49], including the critical arginine residue required for functional folding [50]. Recently, it was suggested that Smac and survivin can bind to each other, an interaction essential for inhibition of Taxolmediated apoptosis in HeLa cells [51]. The key amino acids for this interaction were thought to be the N-terminal AVPI sequence of processed Smac and Asp 71 of survivin. The Asp 71 residue of survivin has previously been shown to be critical for maintaining the acidic surface patch of the BIR domain [49,52,53]. Our results support the suggestion that Smac and survivin can interact and that the N-terminal alanine in the RHG AVPI motif is critical for this. However, the down-regulation of survivin expression in the presence of Smac is only seen at the higher MOI of Ad CMV tSmac transfection suggesting that the interaction may of relatively low affinity. We have shown that the half-life of survivin is markedly reduced in the presence of Smac. The half-life reduction is reversible in the presence of a proteasome inhibitor and the ubiquitination reactions indicate that survivin is targeted for degradation in the presence of Smac. However, it was noted that in vitro survivin ubiquitination was much reduced when recombinant Smac protein was used rather than immunoprecipitate. This implies that other proteins bound to Smac contribute to the survivin degradation indirectly. Curiously, the half-life of survivin was also slightly reduced following transfection with Ad CMV GVPI-tSmac, although the effect was less than with Ad CMV tSmac. The cause of this is uncertain, but it may result from translational inhibition (see below).

Fig. 6. Smac-mediated toxicity occurs independently of Smac/survivin interactions. (A) The 3  104 IGROV1 cells were transfected with 30, 60, or 120 pmol survivin or control siRNA oligonucleotides in 24 well plates. Both total cellular RNA and protein was harvested 72 h later. Following reverse transcription, 100 ng cDNA was subjected to PCR analysis for both survivin and actin and resolved on a 2% agarose gel. Expected bands: survivin 220 bp; actin 230 bp. Thirty micrograms protein was separated on SDS-PAGE gels and analyzed by immunoblot. (B) The 3  104 IGROV1 cells were transfected with Ad CMV tSmac MOI 30 or mock transfected. Six hours later, they were additionally transfected with 30 or 60 pmol survivin or control siRNA oligonucleotides or mock transfected (No RNAi). Cell survival was assessed 66 h later by MTT assay. IGROV1 cells were also transfected with Ad CMV tSmac MOI 30 in the absence ( ) or presence (+) of Ad survivin T34A (T34A) MOI 30 and cell survival assessed 72 h later by MTT assay. Results represent survival relative to cells not transfected with Ad CMV tSmac; mean F SD, n = 4. Protein was also harvested from 30 pmol control RNAi and 30 pmol survivin RNAi-transfected wells and immunoblotted for Smac. (C) The 5  104 IGROV1 cells were transfected with Ad LM-X MOI 30 (X), Ad CMV tSmac (MOI 10 or 30), or mock transfected (M). Six hours later, they were additionally transfected with either 1 Ag pcDNA3 wild-type survivin or 1 Ag of a control plasmid. Cell survival was assessed 66 h later by MTT assay. Protein was also harvested from transfected cells and 10 Ag separated on an SDS-PAGE gel and immunoblotted for Smac and survivin.

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Survivin expression normally peaks at G2/M in the cell cycle with a rapid reduction in G1. This is partially due to the presence of G1 transcriptional repressors elements within the survivin promoter [54], but we have also shown that cell cycle-dependent ubiquitination and degradation in the proteasome pathway represent major posttranslational controls on the levels of survivin in 293 cells [39]. We do not believe that alterations in the cell cycle following transfection with Ad CMV tSmac explain the reduction in survivin expression seen here: The cell cycle analysis in Fig. 1B indicates a relative increase in G2/M and a relative reduction in G1, changes which would act to increase the levels of survivin rather than decrease them. Interestingly, we did find that the half-life of survivin in ovarian carcinoma cells is far longer (6 h) than in 293 cells (30 min). Increased survivin stability has also been noted in nonsmall cell lung carcinoma cells and has been attributed to overexpression of cyclooxygenase-2 [55], which is also observed in ovarian carcinoma [56]. Previous studies on the Drosophila proteins, Reaper, Hid, and Grim, suggest that they can promote degradation of DIAP1 by promoting ubiquitination as well as a generalized translational inhibition [57–59]. However, it has recently been shown that Grim, but not Smac, can promote global ubiquitination and specific degradation of XIAP in 293T cells [22]. Our results do not specifically contradict this: We too found that XIAP half-life appeared unaltered in the presence of Smac in ovarian carcinoma cells while the effect of Grim and Smac/DIABLO on survivin was not studied by Silke et al. [22]. Having shown that Smac and survivin can interact in ovarian carcinoma cells, we then sought to investigate whether survivin played an inhibitory role in Smacmediated apoptosis and whether down-regulation of survivin was the primary basis of Smac-induced apoptosis. The results of RNAi experiments demonstrate clearly that survivin does not play such a role in these cells. This is reiterated by the use of the dominant-negative phosphorylation mutant, survivin T34A, which similarly has no effect upon Smac-induced apoptosis, as does overexpression of wild-type survivin. We also observed that survivin RNAi alone did not significantly affect ovarian carcinoma survival compared to control RNAi in the time period of this experiment, although it remains possible that cell survival would have been affected with longer incubation periods. Taken together, these results support the notion that survivin is not primarily an antiapoptotic protein within the same pathway as Smac. There may be certain specific circumstances in which survivin can act to prevent apoptosis, namely, in the response to paclitaxel (Taxol), a chemotherapy drug that functions partially by stabilizing microtubules, when survivin induces a sustained and stable checkpoint arrest [30,60]. Although survivin can influence paclitaxel-induced apoptosis, it does so as a chromosomal passenger protein and not a true Inhibitor of apoptosis.

Finally, we have demonstrated that adenoviral delivery of the processed Smac gene to nude mice bearing ovarian carcinoma xenografts can produce significant increases in survival. As has been noted previously [61], intraperitoneal injection of E1-deleted adenoviral vectors can produce small survival benefits in nude mice. This may be due to nonspecific inflammatory effects of adenoviral proteins or due to low level replication that has been demonstrated in tumor cells infected with E1-deleted viruses, possibly due to abnormalities in G2/M cell cycle checkpoint in malignant cells [62]. However, the presence of the Smac gene increased survival over both control virus and PBS-treated mice and this difference was most marked when the virus was administered in a daily regime. In conclusion, we have shown that adenoviral delivery of the mature processed form of Smac/DIABLO in ovarian carcinoma cells can induce apoptosis. Most importantly, Smac does not cause apoptosis in normal ovarian surface epithelial cells, which is of great significance in the development of Smac as a novel therapy for ovarian cancer. We show that Smac interacts with both XIAP and survivin and acts to down-regulate survivin via ubiquitination and proteasomal degradation. However, we show through RNA interference that survivin appears to play no inhibitory role in Smac-mediated apoptosis. Finally, and very encouragingly, in vivo treatment with adenoviruses encoding processed Smac in two different treatment regimes significantly prolongs the survival of nude mice bearing intraperitoneal ovarian tumors, and we believe that Smac-based therapies have great potential in the treatment of human ovarian carcinoma.

Materials and methods Cell culture and cell viability assays IGROV1 (donated by Dr. M. Ford, Glaxo-Wellcome Research and Development, Stevenage, UK) and OVCAR4 (obtained from R. Camalier, NCI-Frederick, MD, USA) were incubated at 378C with 10% CO2 in air. IGROV1 cells were maintained in DMEM plus 10% heat-inactivated fetal calf serum (FCS), OVCAR4 were maintained in RPMI medium plus 10% FCS. IOSE397 cells were kindly provided by Dr. Nelly Auersberg, University of British Columbia, Canada, and were maintained in DMEM plus 10% FCS supplemented with 1 mg/ ml gentamicin. For cell viability assays, 105 cells were trypsinized and plated into each well of a 24-well plate. The next day, medium was removed and adenovirus added in serum-free medium. After 90 min with gentle rocking, cells were refed with medium +5% FCS. Cell viability was assayed 72 h later by MTT assay [63]. All cell viability assays were performed in quadruplicate and experiments were all performed at least twice. Representative results are shown unless stated.

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Construction of plasmids and recombinant adenoviral vectors The construction of the plasmid pcDNA3 wild-type survivin, the adenoviral vectors Ad LM-X and Ad CMV survivin T34A, and the generation of both full-length Smac cDNA and the plasmid pcDNA3-Smac have all been described elsewhere [37,64,65]. Processed, truncated Smac (tSmac) was generated from pcDNA3-Smac by PCR using the following primers: Sense: 5V-accatggcggttcctattgcacagaaatcagagcc-3V (Kozak consensus sequence in bold, start codon italicized) and antisense: 5V-tttcaatcctcacgcaggtaggcctcc-3V, and the resulting 561 bp fragment was cloned in pcDNA3.1. GVPI-tSmac was generated from pcDNA3.1tSmac by site-directed mutagenesis using the following PCR primers: Sense: 5V-ggaattgcccttaccatgggggttcctattgc-3V (glycine codon italicized) and antisense: 5V-gcaataggaacccccatcgtaagggcaattcc-3V. Both truncated Smac and GVPItSmac were ligated as KpnI/XhoI fragments into the adenoviral plasmid pShuttle-CMV [66]. Ad CMV GFP was generated using the adenoviral plasmid pAdTrackCMV. Recombinant adenoviral particles were geneQ rated according to the provider’s instructions (http://www. coloncancer.org/adeasy.htm). pcDNA3 Tap-Survivin was generated by ligating survivin cDNA as an EcoRI/HindIII fragment into pcDNA3-Tap (kindly provided by Dr. T. Tenev, ICR, London). Flow cytometry The 106 cells were trypsinized 48 h after adenovirus transfection, washed twice with cold PBS, and fixed in 70% ethanol at 48C for at least 30 min. They were then washed twice with phosphate citrate buffer (192 AM Na2HPO4, 40 AM citric acid), treated with RNase A, and stained with propidium iodide. Cell cycle status was analyzed using a FACScalibur flow cytometer (Becton Dickinson). Generation of cytosolic and mitochondria-enriched cellular fractions Subcellular fractions were generated as previously described [37,67]. Briefly, approximately 5  106 cells were washed twice in ice-cold PBS and resuspended in 400 Al icecold buffer A (20 mM Hepes pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 1 Ag/ml each of leupeptin, aprotinin and pepstatin A, and 250 mM sucrose). Cells were lysed on ice for 1 h and then passed five times through a 25-G needle. The lysates were then spun at 15,000  g for 15 min at 48C, after which the supernatant was spun at 105,000  g for a further 20 min at 48C. The pellet from the first spin represented the mitochondriaenriched fraction and the supernatant from the second spin represented the cytosolic fraction. Ten micrograms of each fraction was electrophoresed on 12% SDS-polyacrylamide

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gels. The primary antibody used to detect cytochrome c was a purified mouse IgG2b raised against pigeon cytochrome c (PharMingen Europe, Oxford, UK). Semiquantitative real-time PCR The 3  106 cells were seeded overnight in 10 cm plates before transfection. Forty-eight hours after transfection, total RNA was extracted using RNAzol B (Biogenesis, Poole, UK). The RNA was subjected to DNase treatment before final column purification using the RNEasy RNA purification kit (Qiagen, Crawley, UK). First-strand cDNA was reverse transcribed from the total RNA using the firststrand cDNA synthesis kit (Roche, Indianapolis, IN, USA) using random hexanucleotide primers. One hundred nanograms of cDNA was used in real-time PCR reactions using an ABI Prism 7700 PCR machine. Primers used were as follows; Survivin forward (F): 5V-CCCATAGAGGAACATAAAAAGCATTC-3V; survivin reverse (R): 5V-TCAAAAATTCACCAAGGGTTAATTCT-3V. XIAP F: 5V-GACAGTATGCAAGATGAGTCAAGTCA-3V; XIAP R: 5V-GCAAAGCTTCTCCTCTTGCAG-3V; c-IAP1 F: 5V-TGTTGTCAACTTCAGATACCACTGG-3V; c-IAP1 R: 5V-CATCATGACAGCATCTTCTGAAGA-3V; Smac F: 5V-GCTGGAAACCACTTGGATGACT-3V; Smac R: 5V-TGCGCCAGTTTGATATGCA-3V; 18S F: 5V-CGCCGCTAGAGGTGAAATTC-3V; 18S R: 5V-CATTCTTGGCAAATGCTTTCG-3V. Data were analyzed using the ABI Prism 7700 SDS applications F software and associated data analysis package. Western blots, immunoprecipitation, and TAP purification Following adenoviral transfection, 106 cells were scraped into 100 Al lysis buffer (150 mm NaCl, 50 mM Tris pH 7.5, 0.05% SDS, 1% Triton X100) and sonicated on ice. Twenty to 30 Ag protein was electrophoresed on SDS-polyacrylamide gels and transferred onto a nitrocellulose filter by semidry blotting. Primary antibodies used were a purified rabbit polyclonal antiserum raised against human Smac (kindly provided by Dr. J. Downward, Cancer Research UK, London, UK), a rat monoclonal anti-Smac/DIABLO IgG2A, a mouse monoclonal anti-human caspase-3 IgG1, a purified rabbit antiserum raised against human caspase-9 (all Alexis Biochemicals, Nottingham, UK), a monoclonal mouse antihuman caspase-8 IgG1 (PharMingen Europe), a mouse monoclonal anti-human XIAP IgG1 (Transduction Laboratories, Lexington, KY, USA), and purified rabbit antisera raised against human survivin, human cIAP-1, and cIAP-2 (all R&D systems, UK). Antibody binding was visualized using ECL (Amersham Pharmacia, Bucks, UK). For immunoprecipitation, 5  106 cells were lysed for 1 h in nondenaturing lysis buffer (1% Triton-X100, 50 mM Tris pH 7.4, 300 mM NaCl, 5 mM EDTA, 10 mM iodoacetamide, 1 mM PMSF, 2 Ag/ml protease inhibitor cocktail), precleared for 60 min with 50 Al normal rat serum/50% protein Gsepharose at 48C and precipitated for 2 h at 48C with 2.5 Ag

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of the rat monoclonal anti-Smac/DIABLO IgG2A conjugated to 50% protein G-sepharose. The precipitate was washed three times in lysis buffer and eluted with 20 Al elution buffer (1% SDS, 100 mM Tris pH 7.4, 10 mM DTT) before resuspension in 50 Al lysis buffer. For Tap purification, cells were lysed on ice in 1% Triton buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton-X100, and 2 Ag/ml protease inhibitor cocktail) for 40 min and then purified as previously described [38]. Intensity of protein expression was quantified using a Typhoon 8600 phosphoimager (Amersham) with Image Quant 5.1 software (Molecular Dynamics, CA, USA). Ubiquitination assays and RNA interference In vitro ubiquitination reactions were performed as previously described [68,69]. Five microliters of purified Tap-survivin and either 2 Al of Smac immunoprecipitate or 2.6 Ag recombinant His-tagged human Smac (R&D Systems, UK) were incubated for 90 min at 308C with 250 nM ubiquitin activating enzyme E1, 2 AM ubiquitin conjugating enzyme 5B, 100 AM His6-tagged ubiquitin, and 2 mM ATP in reaction buffer (50 mM Tris–HCl pH 7.5, 2.5 mM MgCl2, 0.05% NP40, 0.5 mM DTT). Twenty microliters was resolved on a 10% SDS-PAGE gel and blotted with a mouse anti-ubiquitin mAb (PharMingen Europe). For in vivo ubiquitination assays, OVCAR4 cells were transfected with either Ad LM-X or Ad CMV tSmac (both MOI 30), followed 6 h later by transient transfection with pcDNA3-survivin. Twenty-four hours later, cells were refed with medium with or without 12.5 AM lactacystin and harvested 16 h later into 1% Triton lysis buffer. After shearing DNA by repeated passage via a 21-G needle and centrifugation, supernatants were subjected to SDS-PAGE electrophoresis and immunoblotted for survivin. RNAi was performed as previously described [60]. The 3  104 IGROV1 cells growing in antibiotic-free medium in 24 well plates were transfected with Ad CMV tSmac as above. Six hours later, they were transfected with 30–120 pmol of control or survivin siRNA oligonucleotides in OligoFectamine (Invitrogen). Sixty-six hours thereafter, cells were either harvested for RT-PCR, immunoblotting, or assessed for cell survival by MTT assay. In vivo analyses Up to 5  106 IGROV1 cells were injected intraperitoneally into adult female nude mice on day 1. Intraperitoneal virus injections (volume 100 Al per injection) commenced on day 4. Mice were then assessed daily for weight, general health, and accumulation of ascites. When animals were judged to be terminally sick, they were sacrificed, tumors were dissected and weighed, and ascites volume measured. A full pathological assessment was made for each animal.

Acknowledgments This research was funded by Cancer Research UK. We would like to thank Dr. Nelly Auersberg for access to the Canadian Ovarian Tissue Bank and Derek Davies for performing the FACS analyses.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr. 2004.08.029.

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