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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Gene Ther Mol Biol. Author manuscript; available in PMC 2010 April 26.
Published in final edited form as:
Gene Ther Mol Biol. 2009; 13B: 264–273.
PMCID: PMC2859835
NIHMSID: NIHMS168710

Inhibition of caspase-3 by Survivin prevents Wee1 Kinase degradation and promotes cell survival by maintaining phosphorylation of p34Cdc2

Summary

The anti-apoptotic protein Survivin and the cyclin-dependent kinase p34Cdc2 regulate cell cycle progression and apoptosis. p34Cdc2 activation is required for its pro-apoptotic activity and phosphorylation of p34Cdc2 at Tyrosine-15 (Tyr15) maintains p34Cdc2 in an inactive state. In BaF3 IL-3-dependent murine hematopoietic cells, over-expression of wild-type (wt)-Survivin increased Tyrosine phosphorylation of p34Cdc2, while over-expression of dominant-negative (dn) T34A-Survivin decreased Tyr15 phosphorylation. The increased phospho-Tyr15 levels associated with ectopic wt-Survivin directly correlated with enhanced BaF3 cell survival upon growth factor withdrawal, while conversely, low phospho-Tyr15 levels and decreased survival were seen in BaF3 cells expressing ectopic dn-Survivin. Tyrosine-15 phosphorylation of p34Cdc2 is mediated by the Wee1 Kinase, a known target of caspase-3. In BaF3 cells over-expressing wt-Survivin, 2-fold higher levels of Wee1 protein were detected compared to cells expressing vector or dn-Survivin. Treatment of control vector-transduced BaF3 cells with the selective caspase-3 inhibitor Ac-DEVD-CHO increased p34Cdc2-Tyr15 phosphorylation and Wee1 protein levels. In a similar fashion, over-expression of wt-Survivin maintained high levels of phospho-Tyr15-p34Cdc2 and Wee1 protein. Since Survivin requires Hsp90 for stability, we treated cells with the Hsp90 inhibitors AICAR and 17-AAG to further link Survivin to blocking p34Cdc2 activation. Treatment of BaF3 cells expressing ectopic wt-Survivin with AICAR or 17-AAG significantly reduced p34Cdc2-Tyr15 phosphorylation compared to vehicle-treated controls. These results suggest that Survivin protects the p34Cdc2-Tyr15-targeting kinase Wee1 from degradation by blocking caspase-3 activation leading to inhibition of the pro-apoptotic function of p34Cdc2 and enhanced cell survival.

Keywords: Survivin, p34Cdc2, Wee1, caspase-3, Apoptosis

I. Introduction

Survivin is a member of the inhibitor of apoptosis protein family characterized by the baculovirus inhibitor of apoptosis repeat (BIR) domain that confers resistance to apoptosis (Ambrosini et al. 1997). Survivin was first identified as a protein highly expressed in all cancers and in fetal tissues but absent in most adult differentiated tissues (Ambrosini et al. 1997; Adida et al. 1998). It was later discovered to be present in non-terminally differentiated adult tissues such as hematopoietic stem cells (Fukuda and Pelus 2001; Fukuda et al. 2002), T-cells (Fukuda and Pelus 2001; Kornacker et al. 2001) and in keratinocyte (Marconi et al. 2007) and neural (Jiang et al. 2005) stem cells. In hematopoietic stem and progenitor cells, Survivin plays a role in both apoptosis and regulation of cell cycle (Li et al. 1998), through the inhibition of pro-apoptotic proteases caspases 3, 7 (Tamm et al. 1998) and 9 (Chandele et al. 2004), as well as other pro-apoptotic factors such as Smac/Diablo (Song et al. 2003). Survivin is up-regulated by hematopoietic growth factors and in hematopoietic cells is required for cell cycle entry and cell cycle progression (Fukuda et al. 2002; Fukuda and Pelus 2002). At anaphase, Survivin is phosphorylated by p34Cdc2/Cyclin B1 kinase, which helps guide cells through mitosis (O’Connor et al. 2000; Fortugno et al. 2002). Survivin helps localize Aurora Kinase to the head of the mitotic spindle where it also inhibits the activation of caspase 9, thereby avoiding mitotic catastrophe (O’Connor et al. 2000; Uren et al. 2000; Wheatley et al. 2001; Bolton et al. 2002). Survivin gene knockout is embryonic lethal (Uren et al. 2000) attesting to its critical function.

Cyclin-dependent kinase 1 (Cdk1), also known as cell division cycle-2 gene (p34Cdc2) together with Cyclin B1 forms a heterodimeric kinase (Izumi and Maller 1991). In cell cycle, p34Cdc2/Cyclin B1 is thought of as a master regulator, ushering cells from G2 into M phase (Draetta and Beach 1988; Draetta et al. 1989; Morla et al. 1989). In addition to its role in cell cycle, p34Cdc2 activation is required for cells to undergo apoptosis (Meikrantz et al. 1994; Shi et al. 1994; Chen et al. 1995; Shi et al. 1996; Yao et al. 1996). The p34Cdc2/Cyclin B kinase phosphorylates Survivin on Threonine-34, which is considered to be a stabilizing event required for Survivin’s anti-apoptotic effect (O’Connor et al. 2000).

Activated caspases mediate apoptosis through degradation of a variety of proteins and cellular processes. Recently, studies have shown that the p34Cdc2-targeting Wee1 kinase contains a caspase-3 cleavage site (Zhou et al. 1998; Riedl and Salvesen 2007). Wee1 kinase is one of two members of a family of kinases that phosphorylate p34Cdc2 at Tyrosine-15, which inactivates the pro-apoptotic function of p34Cdc2 (Mueller et al. 1995; Fattaey and Booher 1997). Wee1 kinase has also been shown to have anti-apoptotic effects when over-expressed in combination with Stem Cell Factor in CD34+ umbilical cord blood cells challenged with cytotoxic chemotherapeutic agents (Lei et al. 2007) and can protect BHK cells against granzyme-B induced apoptosis (Chen et al. 1995). It is becoming clear that one of the major mechanisms for Wee1-mediated protection from apoptosis is through phosphorylation and inactivation of p34Cdc2 (Heald et al. 1993; Chen et al. 1995).

We found that ectopic Survivin elevated phospho-Tyrosine-15 (pTyr15) p34Cdc2 levels and protected cells from apoptosis. Since Survivin does not contain intrinsic kinase activity, and both Survivin and Wee1 are associated with resistance to apoptosis, we evaluated whether the mechanism of action of Survivin phosphorylation and inactivation of p34Cdc2 was mediated through Wee1. Our results show that Survivin and Wee1 do not interact directly, but that Survivin protects Wee1 kinase from proteosomal degradation through inhibition of caspase-3 activity, resulting in elevated levels of Wee1 kinase protein that maintains inhibitory inactivating Tyr-15 phosphorylation of p34Cdc2, blocking its pro-apoptotic activity.

II. Materials & methods

A. Cell lines

Murine BaF3 IL-3-dependent hematopoietic cells were transduced with wild-type (wt) or dominant-negative (dn), Threonine-34-to-Alanine (T34A) mutation Survivin constructs in the bicistronic MIEG3(−) eGFP containing vector as previously described (Wang et al. 2004). GFP positive cells were FACS-sorted, gating on the highest luminescence-emitting cells (high eGFP expression correlates directly with Survivin expression) and transduced cells maintained as cell lines. BaF3 cells were cultured in RPMI-1640 (Lonza Inc., Allendale, NJ) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) (HyClone Sterile Systems, Logan, UT) and 100 pg/mL of recombinant murine IL-3 and 100 units/mL of Penicillin/Streptomycin (Lonza Inc.) in T75 tissue culture flasks.

The human MCF-7 breast cancer epithelial cell line was cultured in MEMα media supplemented with 10% HI-FBS, 100 units/mL of Penicillin/Streptomycin and 5 μL/mL L-Glutamine (200mM) (Sigma-Aldrich, St Louis, MO) in 10 cm tissue culture dishes. Human Jurkat T-cells were cultured in RPMI-1640 media supplemented with 10% HI-FBS and 0.1% sterile 2-mercaptoethanol (Bio-Rad Laboratories, Hercules, CA) in T75 tissue culture flasks.

B Antibodies and Reagents

The caspase inhibitors Ac-DEVD-CHO, Ac-LEHD-CHO and Z-VAD-FMK and the Hsp90 inhibitor AICAR were purchased from BioMol International (Plymouth Meeting, PA). The anti-Wee1, anti-p34Cdc2 and anti-actin antibodies were purchased from Santa Cruz Biotechnology (La Jolla, CA). Anti-p34Cdc2 phospho-Tyrosine-15, anti-p34Cdc2 and anti-PARP antibodies were from Cell Signaling (Boston, MA). Anti-Survivin antibody (clone AF886) was from R&D Systems (Minneapolis, MN). The Annexin-V-PE and 7-AAD were purchased from BD Pharmingen (San Jose, CA).

C. Cell Lysis and Immunoblotting

Cell lysates were prepared using Triton-X lysis buffer (50mM HEPES, pH 7.0, 150mM NaCl, 10% Glycerol, 1.2% Triton X-100, 1mM EGTA, 1mM EDTA, 10mM sodium pyrophosphate, 100mM NaF, 1mM phenylmethylsulfonyl fluoride, 1mM sodium orthovanadate) or Urea-SDS lysis buffer (62.5 mM Tris (pH 6.8), 6 M urea, 10% glycerol, 2% SDS, 0.003% bromophenol blue, and 5% 2-mercaptoethanol) (Panvichian et al. 1998) supplemented with Complete Mini protease inhibitor cocktail (Roche Diagnostics Inc., Indianapolis, IN). The protein content in cell lysates was determined using a Bradford assay (Bio-Rad Laboratories, Hercules, CA). Lysates were heated at 100 °C for 5 minutes then run on SDS-PAGE gels (8% or 15%) and transferred to PVDF membranes (Millipore, Billerica, MA) at 110 milliamps for 2 hours. The membranes were blocked with 5% milk solution for 1 hour then probed for various proteins. Stripping and reprobing was performed using Restore western blot stripping reagent (Pierce Biotechnology, Rockford, IL).

D. Immunoprecipitations

Human Jurkat cells were harvested in log-phase growth and total cell lysates prepared as described above. Two and one-half mg of lysate for each of three immunoprecipitations, normal rabbit IgG, Survivin and Wee1, were pre-cleared using 20 μL per sample of protein agarose A/G beads (Santa Cruz Biotechnology) for 15 minutes with rocking at 4 °C and pre-cleared lysates transferred to new tubes containing 2.5 μg of the same antibodies used for immunoprecipitation. Lysates were incubated for one hour and 50 μL of protein agarose A/G beads added to each sample and incubated overnight with rocking at 4 °C. Immunoprecipitates were subsequently washed three times using Triton-X lysis buffer and 35 uL of SDS-PAGE loading buffer added to immunoprecipitated samples. Immunoprecipitates and total cell lysate samples were heated at 100 °C for 5 minutes and run on SDS-PAGE gels.

E. Treatments

Log-phase growing BaF3 cells were counted, washed and resuspended in serum-free RPMI-1640 and plated in 6-well tissue culture plates at a concentration of 1×106 cells per mL in 4 mL of media two-hours prior to treatment. Caspase inhibitors were prepared as 1000X stock solutions in DMSO (Ac-DEVD-CHO and Ac-LEHD-CHO at 25 mM and Z-VAD-FMK at 20 mM). Control groups received DMSO alone. Cells were treated for 2 hours at 37 °C, harvested and lysates prepared. For analysis of the effects of Hsp90 inhibition, AICAR was prepared in DMSO and used at 25 μM. Cells receiving AICAR or DMSO were harvested at various time points, lysates prepared using Triton-X lysis buffer and samples run on SDS-PAGE. In order to measure apoptosis, BaF3 cells were either plated at 1×105 cells/mL in 4 mL of normal growth media or RPMI with 1% FBS without IL-3 for 0, 24 or 48 hours, cell pellets collected, stained with Annexin V and 7-AAD and analyzed by flow cytometry. Adherent MCF7 cells were treated with caspase inhibitors in similar fashion to BaF3 cells. MCF7 cells were incubated for 5 minutes with 1 mL of trypsin (Sigma) at 37 °C, counted and plated at 2.5 × 105 cells per mL in 10 mL of media within a 10 cm tissue culture plates for 24 hours prior to treatment to allow readherence to the plate after trypsinization. At 24 hours post-plating, cells were treated and then harvested using trypsin, washed and lysates prepared using either Triton-X or Urea-SDS lysis buffer.

F. Transient transfection

Human wild-type Survivin and T34A dn-Survivin in MIEG-IRES-EGFP plasmid were prepared as previously described (Fukuda et al. 2002; Wang et al. 2004). Transient transfection of MCF7 using MIEG vectors containing Survivin cDNAs was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Empty plasmid was used as a control. Transfection efficiencies of ≥30% were routinely observed by flow cytometry based on GFP expression. After transfection, cells were cultured for 24 hours, trypsinized, washed and lysates prepared as described.

G. Flow Cytometry

In order to quantitate apoptosis, cells were washed and resuspended in Annexin-V binding buffer and Annexin-V and 7-AAD (BD Pharmingen) added to individual samples. Annexin-V single-positive, 7-AAD single-positive, Annexin-V/7-AAD double-positive and double-negative populations were quantitated in triplicate samples by FACS using BD software CellQuest.

III. Results

A. Over-expression of Survivin correlates with survival and elevated phosphoTyr15 p34Cdc2

As we have previously shown, ectopic Survivin protects BaF3 cells from growth factor withdrawal-induced apoptosis and enhances overall cell growth (Wang et al. 2004), while Survivin disruption produced by over-expression of the T34A dn-Survivin construct increases the number of Annexin V+ cells and reduces viable cell growth (Figure 1A, 1B). Analysis of BaF3 cells expressing wt- or dn-Survivin by western blot demonstrates that cells expressing ectopic Survivin contain ~2-fold higher levels of Tyrosine-15 phosphorylated p34Cdc2 (Figure 1C). In cells in which Survivin is disrupted by T34A Survivin, p34Cdc2 tyrosine phosphorylation levels were ~4-fold lower than in cells expressing ectopic Survivin. These findings are consistent with findings by others that indicate that p34Cdc2 activation (dephosphorylation) is required for inhibition of apoptosis (Meikrantz et al. 1994; Shi et al. 1994; Chen et al. 1995; Shi et al. 1996; Yao et al. 1996) and correlate directly with enhanced apoptosis and reduced cell viability observed in our culture experiments. While it is known that p34Cdc2 can phosphorylate Survivin, Survivin does not contain intrinsic kinase activity and therefore cannot directly phosphorylate p34Cdc2. Since the Wee1 kinase can phosphorylate p34Cdc2 on Tyrosine-15, we probed lysates from BaF3 cells expressing wt- or dn-T34A Survivin for Wee1. Cells over-expressing Survivin showed significantly higher levels of Wee1 protein compared to controls (Figure 1C). No Wee1 protein was detected in cells expressing T34A-Survivin. These results directly correlated with p34Cdc2 phosphorylation and suggested that Wee1 and Survivin may be functionally linked.

Figure 1
Effects of Survivin Modulation on Apoptosis, Cell Viability and Protein Levels of p34Cdc2 and Wee1

B. WT-Survivin over-expression is sufficient to maintain Wee1 levels

To investigate the mechanism whereby Survivin increases Wee1 kinase protein levels, we first determined if there was direct binding between Survivin and Wee1. Endogenous co-immunoprecipitation experiments were performed using Jurkat T-cells that express high endogenous levels of both Wee1 and Survivin. While both Survivin and Wee1 proteins could each be independently precipitated and detected using specific antibodies, no direct binding between Wee1 and Survivin was observed (Figure 2A). Since a recent report indicated that Wee1 is a target of caspase-3 (Zhou et al. 1998; Alam et al. 1999) and Survivin can inhibit caspase-3 (Tamm et al. 1998), we evaluated if the elevated Wee1 protein detected in cells over-expressing wt-Survivin was a result of protection of Wee1 from caspase-mediated degradation. Treatment of MIEG- or wt-Survivin-transduced BaF3 cells with 50 μM of the specific caspase-3 inhibitor Ac-DEVD-CHO for 2 hours followed by western blotting showed that the levels of pTyr15- p34Cdc2 were increased (4-fold) in MIEG-transduced cells in the presence of the caspase-3 inhibitor (Figure 2B and 2C). In contrast, levels of pTyr15-p34Cdc2 were unaffected by pharmacological caspase inhibition in wt-Survivin over-expressing cells. Reduction in the levels of cleaved poly(ADP-Ribose) polymerase (PARP), a known caspase-3 selective target, verified that Ac-DEVD-CHO effectively reduced caspase-3 activity (Figure 2B, 2C).

Figure 2
Survivin Maintains Tyrosine-15 Phosphorylation of p34Cdc2

In wt-Survivin BaF3 cells, levels of cleaved PARP were significantly reduced compared to controls and were further reduced in the presence of Ac-DEVD-CHO, although this did not lead to greater levels of pTyr15-p34Cdc2.

In order to determine if increased p34Cdc2-Tyr15 phosphorylation was due solely to inhibition of caspase-3, additional studies were performed using the selective caspase 7 inhibitor Ac-LEHD-CHO and the pan-caspase inhibitor Z-VAD-FMK. In control MIEG-transduced cells, all three caspase inhibitors increased the levels of phosphorylated Tyrosine-15 p34Cdc2 (1.3–1.6 fold) with no effect on total p34Cdc2. The highest levels of pTyr15 p34Cdc2 were seen in cells treated with the pan-caspase inhibitor Z-Vad-FMK. In cells over-expressing wt-Survivin, ectopic Survivin was sufficient to maintain elevated pTyr15 p34Cdc2 levels that could not be increased further in the presence of any of these pharmacological caspase inhibitors. Total Wee1 level varied slightly with the different caspase inhibitors. In control MIEG treated cells an increase in Wee1 protein directly correlated with increased levels of pTyr15-p34Cdc2 observed in the presence of the caspase inhibitors. In wt-Survivin over-expressing cells Wee1 levels were higher than the levels in the MIEG-transduced cells except for vector cells treated with the pan-caspase inhibitor Z-Vad-FMK (Figure 2D) and could not be increased further in the presence of the caspase inhibitors. Though varying slightly, levels of Wee1 protein stayed at or above 2-fold over vector treated control levels.

To further demonstrate that the efects of wt-Survivin and caspase inhibition observed in BaF3 cells and MCF7 cells are specific to caspase-3, MCF7 cells were transiently transfected with wt-Survivin and dn-Survivin cDNAs and Wee1 protein levels and Tyr15 p34Cdc2 phosphorylation evaluated (Figure 4). No effect on total Wee1 protein, total Cdc2 or phospho Tyr15 Cdc2 levels was observed in MCF7 cells over expressing wt-Survivin or expressing ectopic dn-Survivin, strongly suggesting that selective inhibition of caspase -3 responsible for the effects of Survivin on Wee1 protein and Tyr15 Cdc2 phosphorylation. In addition, no change in the caspase-3 selective target PARP was observed.

Figure 4
Modulation of Survin levels in MCF7 Cells has no effect on Wee1 Protein Level and Tyrosine-15 Phosphorylation of p34Cdc2

D. Destabilization of Survivin Abrogates the Effect on Wee1

Survivin protein is stabilized by interacting with the heat shock protein Hsp90 (Fortugno et al. 2003; Plescia et al. 2005; Meli et al. 2006). We therefore evaluated whether blocking Survivin-Hsp90 interaction would block increased p34Cdc2 phosphorylation to further confirm a specific role for Survivin in increasing p34Cdc2 phosphorylation and Wee1 protein levels. BaF3 cells over-expressing wt-Survivin were treated with the Hsp90inhibitor AICAR or DMSO for 1, 2, 5 or 6 hours and lysates analyzed for Tyr15 p34Cdc2 phosphorylation levels. No effect of AICAR on pTyr15 p34Cdc2 levels were observed during the first 2 hours of treatment, however at 5 hours the levels of pTyr15 p34Cdc2 in BaF3 cells treated with the Hsp90 inhibitor were significantly lower than untreated cells and the effect was even more pronounced at 6 hours, where essentially no pTyr15 p34Cdc2 was detected (Figure 5A). Consistent with the destabilizing effects of Hsp90 inhibitors on Survivin protein stability, Survivin protein levels were reduced coincident with reduction in pTyr15 p34Cdc2. In similar fashion, treatment of BaF3 cells expressing wt-Survivin with 100 nM 17-AAG, a Geldanamycin-derivative, that blocks the Hsp90 ATP-binding site, resulted in a decrease in Wee1 protein, pTyr15 p34Cdc2, Survivin and whole PARP levels after 24 hours treatment coincident with reduced levels of Hsp90 protein (Figure 5B).

Figure 5
Inhibition of Survivin-Hsp90 Interaction Abrogates the Effect of wt-Survivin on Maintenance of PhosphoTyrosine-15 p34Cdc2 Levels

IV. Discussion

A number of studies show that Survivin over-expression confers resistance to apoptosis that is mediated through the inhibition of pro-apoptotic proteins such as caspases 3, 7 and 9 (Li et al. 1998; Song et al. 2003; Chandele et al. 2004). We have found that Survivin over-expression also increases phosphorylation of p34Cdc2 on Tyrosine-15. Although p34Cdc2 can promote mitosis, it can also mediate apoptosis, and phosphorylation of p34Cdc2 at Tyrosine-15 inactivates p34Cdc2, blocking its pro-apoptotic activity (Meikrantz et al. 1994; Shi et al. 1994; Chen et al. 1995; Shi et al. 1996; Yao et al. 1996). Increased phosphoTyr15 p34Cdc2 is consistent both with the role of p34Cdc2 and the action of the anti-apoptotic protein Survivin. However, since Survivin does not contain intrinsic kinase activity, it is not clear how p34Cdc2 phosphorylation is achieved. Herein, we show that Survivin increases the level of the Wee1 kinase that phosphorylates p34Cdc2 by blocking Wee1 degradation by caspase-3. The increase in Wee1 protein and Tyrosine-15 phosphorylation of p34Cdc2 directly correlate with increased survival in cells expressing ectopic wild-type Survivin. To our knowledge this is the first report demonstrating a functional link between the Wee1 kinase and Survivin.

A functional link between Survivin and p34Cdc2 has been previously reported (O’Connor et al. 2000), however cross-talk between the two proteins has recently come into question. Several reports show a correlation between p34Cdc2 activation and apoptosis (Meikrantz et al. 1994; Shi et al. 1994; Chen et al. 1995; Shi et al. 1996; Yao et al. 1996), thus it makes sense that p34Cdc2 inactivation correlates with Survivin over-expression. However, an association between induction of increased Survivin and an increase in p34Cdc2 activation in response to ethanol in a gastric epithelial cell line model was recently reported (Jones et al. 2008). Although these findings seem contradictory to our findings in normal hematopoietic cells, differences in normal versus transformed cells could be responsible. Also, in the gastric epithelial model it is assumed that active p34Cdc2 results in increased Survivin stabilization and thus the two are up-regulated simultaneously. However, we show that an increase in Survivin expression in turn has a down-regulatory effect on p34Cdc2 activity through Wee1 kinase protection from caspase-3 mediated degradation, thus forming a negative feedback loop resulting in enhanced evasion of apoptosis. Our proposed regulatory role of Survivin and p34Cdc2 is consistent with our previous report that Survivin functions as a regulatory element in the Mdm2-p53 pathway, where it reduces p53 protein and mRNA through protection of Mdm2 from caspase-mediated degradation (Wang et al. 2004).

Although Survivin is associated with inhibition of caspase activity, whether it directly binds and inhibits caspase-3 directly is not clear (Banks et al. 2000; Wright et al. 2000; Shin et al. 2001; Li et al. 2008). Nevertheless, over-expression of wt-Survivin in our model system resulted in an increase in whole PARP levels as compared to cells over-expressing vector alone and PARP cleavage is an established measure of active caspase-3 (Tewari et al. 1995). We have also shown that ectopic Survivin is sufficient to maintain phosphoTyr15 p34Cdc2 levels even in the presence of an exogenous specific caspase-3 inhibitor Ac-DEVD-CHO and that this effect is absent in caspase-3 deficient cells, even in the presence of ectopic wt-Survivin or a dn-Survivin construct. Taken together these results strongly support Survivin-mediated caspase-3 inhibition, however they do not prove whether this represents a direct effect on caspase-3. We also show that Survivin over-expression is sufficient to maintain higher levels of Wee1 kinase compared to vector-control cells even in the presence of caspase inhibition, and present strong evidence that this effect is mediated through caspase-3 inhibition, although we cannot rule out the involvement of other caspases, at least in BaF3 cells. However, the lack of increase in Wee1 and phospho-Tyr15 p34Cdc2 levels in MCF7 cells deficient only in caspase-3 even in the presence of ectopic over expression of wt-Survivin or a dn-Survivin mutant strongly suggests that caspase-3 is the primary caspase mediating protection of Wee1. It has been reported that the Wee1 kinase is a caspase-3 target and is associated with resistance to apoptosis (Chen et al. 1995; Lei et al. 2007), similar to Survivin. Thus our finding that Wee1 kinase protein levels are increased by wt-Survivin over-expression through caspase-3 inhibition is consistent with the mechanism of both proteins.

Caspase-3 has been reported to play a role in the maintenance of the hematopoietic stem cell (HSC) pool (Janzen et al. 2008). HSCs deficient in caspase-3 show an accelerated proliferation and retarded differentiation. This finding is consistent with our past studies that Survivin is up-regulated in response to growth factors in quiescent CD34+ cells and required for their entry into cell cycle (Fukuda and Pelus 2001; Fukuda et al. 2002; Fukuda and Pelus 2002). In addition, using a conditional Survivin knockout mouse model, Survivin deletion results in significant impairment in HSC number and HSC function (Fukuda S. and Pelus, L.M. unpublished). Taken together these studies indicate that Survivin and caspase-3 are intimately involved in HSC function. Increased caspase-3 inhibition by Survivin maintains the stem cell pool and loss of Survivin protein with subsequent increased caspase-3 activity results in loss of HSC function and perhaps increased cell differentiation. Very little is known about the role of Wee1 kinase in hematopoiesis and it will be interesting to determine how it is regulated in HSCs and in their differentiated progeny. Since Survivin is up-regulated endogenously in HSCs and initiates proliferation, our present results suggest that regulation of Wee1 protein in these cells is likely directly correlated with Survivin levels. In addition, reports have shown that regulation of Wee1 correlates with cell survival (Heald et al. 1993; Chen et al. 1995; Leach et al. 1998; Wang et al. 2005; Lei et al. 2007). Thus, evaluation of whether inhibition of Wee1 kinase in Survivin-over-expressing cancers would down-regulate inhibition of apoptosis could identify a novel and effective chemotherapeutic strategy.

In summary, our results indicate that Survivin over-expression mediates protection of the Wee1 kinase through inhibition of caspase-3, which in turn leads to an increase in phosphoTyr15 p34Cdc2. In the absence of Survivin, caspase-3 is not inhibited, Wee1 is degraded and p34Cdc2 is activated or kept active and apoptosis is initiated. It is possible that other proteins that affect p34Cdc2 activity, particularly p21Cip1/Waf1 that can inhibit p34Cdc2 (Satyanarayana et al. 2008), and is itself also a caspase-3 target (Gervais et al. 1998) and has an important role in the proliferation of normal HSCs by Survivin (Fukuda et al. 2004), may play a role in the anti-apoptotic Survivin-caspase-3-Wee1-p34Cdc2 axis we have described.

Figure 3
Wee1 Protein Level is Maintained in the Absence of Caspase-3 Activity

Acknowledgments

Supported by grants HL079654, HL69669 and HL007910 from the National Institutes of Health

Biography

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Javier Rivera-Guzman, Ph.D

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