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Clinical Cancer Research Vol. 10, 1894-1900, March 2004
© 2004 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Reconstitution of Caspase-3 Confers Low Glucose-Enhanced Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Cytotoxicity and Akt Cleavage

Yong J. Lee1, Christopher J. Froelich2, Naoya Fujita3, Takashi Tsuruo3 and Jin H. Kim1

1 Department of Surgery and Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; 2 Department of Medicine, Northwestern University Medical School, Chicago, Illinois; and 3 Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan

ABSTRACT

Purpose and Experimental Design: We have previously observed that glucose deprivation enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death as well as caspase activation (caspase-3, -9, and -8) in human prostate adenocarcinoma DU-145 cells. In this study, we used caspase-3-deficient MCF-7 breast cancer cells to examine the possible role of caspase-3 in glucose deprivation-enhanced TRAIL cytotoxicity.

Results: Combined glucose deprivation and 200 ng/ml TRAIL treatment markedly induced cytotoxicity in caspase-3 cDNA transfected cells (MCF-7/casp-3) but not in control vector transfected cells (MCF-7/vector). We also observed that the level of Akt, an antiapoptotic protein, was reduced by treatment with TRAIL in MCF-7/casp-3 cells but not in MCF-7/vector cells. The reduction of Akt by TRAIL was promoted in the absence of glucose in MCF-7/casp-3 cells. However, pretreatment with 20 µM Z-LEHD-FMK, a caspase-9 inhibitor, protected MCF-7/casp-3 cells from the combinatorial treatment of TRAIL and glucose deprivation-induced cytotoxicity. This compound also prevented the reduction of Akt level during the combinatorial treatment. Moreover, this Akt reduction was not inhibited by treatment with MG-132, a proteosome inhibitor. Data from site-directed mutagenesis show that Akt was cleaved at amino acid 108, but not 119, during treatment with TRAIL and glucose deprivation.

Conclusions: Our results suggest that caspase-3 is involved in the reduction of Akt level, and its involvement is mediated through caspase-9 activation. The reduction of Akt level is also due to cleavage of Akt rather than degradation of Akt.

Introduction

The abnormalities of the tumor vasculature result in insufficient blood supply and development of tumor microenvironment, which is characterized by regions of fluctuating and chronic hypoxia, low extracellular pH, and low glucose concentrations. Our previous studies have shown that glucose deprivation enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity in DU-145 cells (1) . Our studies have also revealed that the combined treatment of TRAIL with glucose deprivation promotes caspase activation. In this study, we further investigated the role of caspases, in particular caspase-3, in low glucose-enhanced TRAIL cytotoxicity.

Caspase-3 is an effector caspase that is activated by apical caspases (caspase-2, -8, -9, and -10). Interestingly, recent evidence suggests that caspase-3 is essential for procaspase-9 processing (2) . It is also required for DNA fragmentation and membrane blebbing associated with apoptosis (3) . MCF-7, a breast cancer cell line, is caspase-3 deficient (3) and relatively insensitive to many chemotherapeutic agents (4) . However, reconstitution of caspase-3 sensitizes MCF-7 cells to chemotherapeutic agents (4) . It is probably due to the fact that caspase-3 is a key caspase in the apoptotic signaling cascade (5) . Recent studies indeed demonstrate that caspase-3 plays an important role in both death receptor- and mitochondria-mediated apoptosis (6) . This caspase is located in a central position as an integrator and amplifier of apoptotic pathways.

Akt (also known as PKB) is a serine/threonine kinase. It was originally discovered as the cellular counterpart of the v-Akt transforming protein of a retrovirus (AKT8) that caused T-cell lymphomas in mice (7) . Akt is recruited to the plasma membrane and activated by phosphorylation at threonine 308 and serine 473 in response to growth factors (8) . The molecule responsible for the recruitment of Akt is phosphatidylinositol 3'-kinase (8 , 9) . Phosphatidylinositol 3'-kinase consists of a regulatory subunit (p85) that binds to an activated growth factor/cytokine receptor and undergoes phosphorylation, which results in the activation of its catalytic subunit (P110; Ref. 10 ). Phosphatidylinositol 3'-kinase phosphorylates phosphoinositides at the 3' position of the inositol ring, and its major lipid product is phosphatidylinositol 3,4,5-trisphosphate (11) . Phosphatidylinositol 3,4,5-trisphosphate facilitates the recruitment of Akt to the plasma membrane by binding with the pleckstrin homology domain of Akt (11) . Akt is activated by phosphoinositide-dependent kinase-1 through phosphorylation at threonine 308 and serine 473 (8 , 9 , 12) . Activated Akt is involved in phosphorylation of several transcription factors (e.g., nuclear factor {kappa}B, Forkhead, and cAMP-responsive element binding protein; Refs. 13, 14, 15 ), as well as proapoptotic molecules such as Bad and procaspase-9. Phosphorylation of Bad and procaspase-9 results in inactivation of these molecules and inhibits apoptosis (16, 17, 18) . Recent studies reveal that Akt is cleaved by caspases, probably caspase-3 during apoptosis (19 , 20) . Death domain-containing proteins promote the caspase-dependent cleavage of Akt, and the cleavage of Akt contributes to apoptotic death (21) . In this study, we demonstrate that TRAIL in combination with glucose deprivation promotes the caspase-mediated cleavage of Akt, and this event contributes to apoptotic death.

Materials and Methods

Cell Culture and Survival Determination.
MCF-7/vector or MCF-7/casp-3 cell lines were obtained from Dr. C. J. Froelich (Northwestern University, Chicago, IL). They were established by stable transfection of empty vector or caspase-3 cDNA, respectively, and selected with 2 µg/ml puromycin as described previously (4) . All cells were cultured in DMEM medium with 10% fetal bovine serum (HyClone) and 26 mM sodium bicarbonate for monolayer cell culture. The dishes containing cells were kept in a 37°C humidified incubator with a mixture of 95% air and 5% CO2. Two days before the experiment, cells were plated into 60-mm dishes. For trypan blue exclusion assay (22) , trypsinized cells were pelleted and resuspended in 0.2 ml of medium, 0.5 ml of 0.4% trypan blue solution, and 0.3 ml of PBS solution. The samples were mixed thoroughly, incubated at room temperature for 15 min, and examined.

Production of Recombinant TRAIL.
A human TRAIL cDNA fragment (amino acids 114–281) obtained by reverse transcription-PCR was cloned into a pET-23d (Novagen, Madison, WI) plasmid, and expressed protein was purified using the nickel-nitrilo-triacetic acid His-Bind Resin Superflow according to the manufacturer’s instructions (Novagen).

Treatment with Glucose Deprivation and TRAIL.
Cells were rinsed three times with PBS solution, which took approximately 15 min. Cells were treated with glucose-free DMEM medium with 10% dialyzed fetal bovine serum (Life Technologies, Inc., Grand Island, NY). For TRAIL treatment, cells were replaced with fresh medium containing TRAIL (200 ng/ml).

Transfections.
One microgram of the pFLAG-CMV-2 vectors containing WT-Akt, D108A-Akt, D119A-Akt, or D108A+D119A-Akt cDNA (kindly provided by Dr. Naoya Fujita, University of Tokyo, Tokyo, Japan) was transiently transfected into MCF-7/casp-3 cells using the LipofectAMINE method (Life Technologies, Inc.) and incubated for 48 h.

Reagents and Antibodies.
Polyclonal anti-caspase-3 and -9 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Akt from New England Biolabs (Beverly, MA) was used. Monoclonal antibodies were purchased from the following companies: anti-caspase-8 from Upstate Biotechnology (Lake Placid, NY) and anti-poly (ADP-ribose) polymerase (anti-PARP) from Biomol Research Laboratory (Plymouth Meeting, PA). Other chemicals were purchased from Sigma (St. Louis, MO).

Protein Extracts and PAGE.
Cells were lysed with 1x Laemmli lysis buffer [2.4 M glycerol, 0.14 M Tris (pH 6.8), 0.21 M SDS, and 0.3 mM bromphenol blue] and boiled for 10 min. Protein content was measured with bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). The samples are diluted with 1x lysis buffer containing 1.28 M ß-mercaptoethanol, and an equal amount of protein was loaded on 10–12% SDS-polyacrylamide gels. SDS-PAGE analysis was performed according to Laemmli (23) using a Hoefer gel apparatus.

Immunoblot Analysis.
Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. The nitrocellulose membrane was blocked with 7.5% nonfat dry milk in PBS-Tween 20 (0.1%, v/v) at 4°C overnight. The membrane was incubated with anti-PARP, anti-caspase-8, anti-caspase-3, anti-phopho-Akt, or anti-Akt antibody (diluted according to the manufacturer’s instructions) for 1 h. Horseradish peroxidase conjugated antirabbit or antimouse IgG was used as the secondary antibody. Immunoreactive protein was visualized by the chemiluminescence protocol (ECL; Amersham Biosciences, Arlington Heights, IL).

Results

Glucose Deprivation Promotes TRAIL-Induced Cytotoxicity in MCF-7/Caspase-3 Cells.
We have previously reported that glucose deprivation enhances TRAIL-induced apoptosis in human prostate adenocarcinoma DU-145 cells (1) . To examine whether caspase-3 is involved in this process, we used caspase-3-reconstituted MCF-7 cell line (MCF-7/casp-3) and its control vector transfected cell line (MCF-7/vector). Fig. 1Citation shows that glucose deprivation enhanced TRAIL-induced cytotoxicity, which was determined by the trypan blue exclusion assay, in MCF-7/casp-3 cells. For example, approximately 55% of the cells were killed at the dose of 200 ng/ml TRAIL alone for 2 h of exposure. Glucose deprivation significantly enhanced TRAIL-induced cell death in MCF-7/casp-3 cells; more than 85% of the cells were killed after 2 h of incubation with 200 ng/ml TRAIL. Glucose deprivation alone did not cause cytotoxicity within 3 h (data not shown). In contrast to MCF-7/casp-3 cells, TRAIL did not cause significant cytotoxicity in MCF-7/vector cells. However, minimal but significant increase in cytotoxicity was observed when MCF-7/vector cells were treated with TRAIL in combination with glucose deprivation. Thus, our data suggest that glucose deprivation enhances two different types of death: caspase-3-dependent and caspase-3-independent death. Nonetheless, our data clearly demonstrated that glucose deprivation-enhanced TRAIL cytotoxicity in MCF-7/casp-3 cells (caspase-3-dependent cell death) is greater than that in MCF-7/vector cells (caspase-3-independent cell death).



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  		Fig. 1. Cytotoxicity induced by TRAIL alone or in combination with glucose deprivation in MCF-7/vector and MCF-7/casp-3 cells. Cells were treated with 200 ng/ml TRAIL for various times (1–3 h) in the presence or absence of glucose. Cell survival was determined by the trypan blue exclusion assay. {blacksquare}, TRAIL alone in MCF-7/vector cells; {square}, TRAIL alone in MCF-7/casp-3 cells; {blacktriangleup}, TRAIL + glucose deprivation in MCF-7/vector cells; {triangleup}, TRAIL + glucose deprivation in MCF-7/casp-3 cells. Data are a compilation of five separate experiments.

 
Role of Caspase-3 in a Combination of Glucose Deprivation and TRAIL Treatment-Promoted Cleavage of PARP, Activation of Caspases, and Reduction of Akt Level.
To examine whether the combination of TRAIL and glucose deprivation-enhanced cytotoxicity is associated with apoptotic death, PARP cleavage, the hallmark feature of apoptosis, was investigated. Fig. 2ACitation shows that 200 ng/ml TRAIL alone or in combination with glucose deprivation failed to induce PARP cleavage in MCF-7/vector cells. In contrast, 200 ng/ml TRAIL alone caused PARP cleavage in MCF-7/casp-3 cells; PARP (Mr 116,000) was cleaved yielding a characteristic Mr 85,000 fragment in the presence of TRAIL. PARP cleavage was significantly increased with increasing duration of TRAIL treatment and an absence of glucose (Fig. 2A)Citation .



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  		Fig. 2. Role of caspase-3 in TRAIL alone or in combination with glucose deprivation-induced proteolytic cleavage of PARP (A) or activation of caspases (B). MCF-7/vector or MCF-7/casp-3 cells were treated with 200 ng/ml TRAIL for various times (1–2 h) in the presence or absence of glucose. Lysates from equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-PARP, anti-caspase-8, anti-caspase-9, or anti-caspase-3 antibody. Actin, internal standard actin protein. Con, untreated control cells.

 
It is well known that apoptosis induced by TRAIL is characterized by the activation of caspases (24) . To examine whether the effects of glucose deprivation on TRAIL-induced activation of caspases in MCF-7/vector and MCF-7/casp-3 cells are different, cells were exposed to glucose-free medium and/or 200 ng/ml TRAIL for 1–2 h. The activation of caspase-8 was detected by treatment with TRAIL regardless of glucose deprivation in both cell lines (Fig. 2B)Citation . Procaspase-8 (Mr 55,000), the precursor form of caspase-8, was cleaved to the intermediate (Mr 43,000 and 41,000) and active forms (Mr 18,000). Unlike caspase-8, procaspase-9 (Mr 49,000) was cleaved in only MCF-7/casp-3 cells. The cleavage of procaspase-9 was enhanced in the absence of glucose. Similar results were observed in the activation of caspase-3. In MCF-7/casp-3 cells, TRAIL induced proteolytic processing of procaspase-3 into its active form (Mr 12,000), and the combined treatment with TRAIL and glucose deprivation resulted in an increase in caspase-3 activation compared with TRAIL alone (Fig. 2B)Citation . In MCF-7/vector cells, however, caspase-3 itself was not detected.

TRAIL in Combination with Glucose Deprivation Reduces Akt Level.
We previously observed that low glucose-enhanced TRAIL cytotoxicity is mediated through the Akt pathway (1) . To examine the involvement of Akt in the enhancement of apoptosis by 200 ng/ml TRAIL in combination with glucose deprivation, we determined the level of Akt by Western blotting (Fig. 3)Citation . The Akt level had not changed in MCF-7/vector cells treated with TRAIL and/or glucose deprivation relative to untreated control cells. But in MCF-7/casp-3 cells, unlike MCF-7/vector cells, Akt level was decreased by combined treatment with TRAIL and glucose deprivation. The level of Akt (Mr 60,000) was reduced, whereas a Mr 45,000 band appeared during treatment with TRAIL in combination with glucose deprivation in MCF-7/casp-3 cells.



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  		Fig. 3. Effect of TRAIL alone or in combination with glucose deprivation on Akt in MCF-7/vector or MCF-7/casp-3 cells. Cells were treated with 200 ng/ml TRAIL in the presence or absence of glucose for various times (1–2 h). Lysates from equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-Akt antibody. Actin, internal standard actin protein. Con, untreated control cells.

 
To assess the involvement of caspases in the reduction of Akt, MCF-7/casp-3 cells were pretreated with caspase-9 inhibitor, Z-LEHD-FMK (Fig. 4)Citation or caspase-3 inhibitor, Z-DEVD-FMK (Fig. 5)Citation . Fig. 4Citation and Fig. 5Citation show that 20 µM Z-LEHD-FMK or 20 µM Z-DEVD-FMK protected cells from cytotoxicity (Fig. 4ACitation ; Fig. 5ACitation ), activation of caspases (Fig. 4BCitation ; Fig. 5BCitation ), cleavage of PARP (Fig. 4BCitation ; Fig. 5BCitation ), and reduction of Akt (Fig. 4CCitation ; Fig. 5CCitation ) during treatment with TRAIL alone or in combination with glucose deprivation. These results suggest that reduction of Akt is due to cleavage of Akt, which is mediated through activation of caspases, probably caspase-3. We also observed that proteasome inhibitors (MG-132, lactacystin, and proteasome inhibitor I) did not inhibit Akt cleavage during combined treatment with TRAIL and glucose deprivation (data not shown).



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  		Fig. 4. Effect of caspase-9 inhibitor on TRAIL in combination with glucose deprivation-induced cytotoxicity (A), PARP cleavage and caspase activation (B), as well as Akt reduction (C). MCF-7/casp-3 cells were pretreated with 20 µM caspase-9 inhibitor for 30 min followed by treatment with TRAIL alone or in combination with glucose deprivation. A, cell survival was determined by the trypan blue exclusion assay. Data represent two separate experiments (Expt.). B, lysates from equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-PARP antibody or anticaspase antibody as described in Fig. 1Citation and Fig. 2Citation . Actin, internal standard actin protein. Con, untreated control cells. C, lysates from equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-Akt antibody. Actin, internal standard actin protein. Con, untreated control cells.

 


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  		Fig. 5. Effect of caspase-3 inhibitor on TRAIL in combination with glucose deprivation-induced cytotoxicity (A), PARP cleavage and caspase activation (B), as well as Akt reduction (C). MCF-7/casp-3 cells were pretreated with 20 µM caspase-3 inhibitor (Z-DEVD-FMK) for 30 min followed by treatment with TRAIL alone or in combination with glucose deprivation. A, cell survival was determined by the trypan blue exclusion assay. Data represent two separate experiments (Expt.). B, lysates from equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-PARP antibody or anticaspase antibody as described in Fig. 1Citation and Fig. 2Citation . Actin, internal standard actin protein. Con, untreated control cells. C, lysates from equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-Akt antibody. Actin, internal standard actin protein. Con, untreated control cells.

 
Identification of Cleavage Site in Akt Protein.
Caspases are known to cleave the substrates that contain aspartic acid at the P1 position. Caspase-3 is known to preferentially cleave the substrates that contain aspartic acid residues at both P1 and P4 positions (P4 to P1; DXXD in which X may be any amino acid; Refs. 25 and 26 ). Human Akt protein contains 28 aspartic acid residues. However, cleavage of only two potential sites (TVAD108G, and EEMD119F) can generate a Mr 45,000 fragment of Akt. To determine which site is responsible for the cleavage of Akt, MCF-7/casp-3 cells were transfected with three types of Akt mutant plasmids (D108A, D119A, and D108A + D119A). These plasmids contain a Flag-tagged on N-terminal of Akt mutant cDNA. Fig. 6Citation shows that D108A and D108A + D119A of Akt were not cleaved during combined treatment with TRAIL and glucose deprivation. These results suggest that aspartic acid 108, but not aspartic acid 119, is the cleavage site.



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  		Fig. 6. Identification of cleavage site in Akt protein. MCF-7/casp-3 cells were stably transfected with control plasmids (pcDNA3), plasmids containing a FLAG-tagged wild-type Akt cDNA (Wt), single-point mutant at either aspartic acid 108 (D108A) or aspartic acid 119 (D119A), or double-point mutant (D108 + 119A) Akt cDNA. Cells were exposed to either complete medium or glucose-free medium in combination with TRAIL (200 ng/ml) for 2 h. Lysates from equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-FLAG antibody. Actin, internal standard actin protein.

 
Effect of Uncleavable Akt on a Combination of Glucose Deprivation and TRAIL Treatment-Induced Cytotoxicity.
To assess whether Akt cleavage is responsible for low glucose-enhanced TRAIL cytotoxicity, three types of Akt mutant plasmids transfected MCF-7/casp-3 cells were treated with TRAIL in the absence of glucose. Fig. 7Citation shows that D108A and D108A + D119A of Akt but not aspartic acid 119 of Akt transfected cells are resistant to cytotoxicity. These results suggest that expression of uncleavable Akt prevents TRAIL-induced apoptosis and specifically the enhancement of apoptosis by glucose deprivation.



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  		Fig. 7. Effect of Akt mutants on TRAIL in combination with glucose deprivation-induced cytotoxicity. MCF-7/casp-3 cells were stably transfected with plasmids containing a FLAG-tagged wild-type Akt cDNA (Wt), single-point mutant at either aspartic acid 108 (D108A) or aspartic acid 119 (D119A), or double-point mutant (D108 + 119A) Akt cDNA. MCF-7/vector or MCF-7/casp-3 transfectants were treated with TRAIL alone ({square}) or in combination with glucose deprivation ({blacksquare}). Cell survival was determined by the trypan blue exclusion assay. Data are a compilation of five separate experiments (Expt.).

 
Discussion

Caspase-3 reconstituted MCF-7 cell line (MCF-7/casp-3) was useful for studying the specific role of caspase-3-dependent signaling in response to TRAIL in combination with glucose deprivation. As shown in Fig. 1Citation and Fig. 2Citation , caspase-3 reconstitution sensitized MCF-7 cells to TRAIL-induced apoptosis in the absence of glucose. An increase in PARP cleavage and activation of caspase-8, -9, and -3 was also observed after combined treatment with TRAIL and glucose deprivation in MCF-7/casp-3 cells. In contrast, caspase-3-deficient MCF-7 cells (MCF-7/vector) were resistant to TRAIL cytotoxicity (Fig. 1)Citation . Although caspase-8 activation was observed in MCF-7/vector cells, activation of caspase-9 was not detected during treatment with TRAIL alone or in combination with low glucose (Fig. 2)Citation . Our data clearly show that Akt was cleaved in MCF-7/casp-3 but not in MCF-7/vector, even though caspase-8 was activated during treatment with TRAIL in the absence of glucose in both cell lines. These results suggest that caspase-3, but not caspase-8, is responsible for Akt cleavage. Our observations were consistent with previous observations that caspase-8 is an initiator but not an executioner. However, recent studies suggest that inhibition of glucose metabolism enhances TRAIL-induced apoptosis by increased activation of caspase-8 (27) . Obviously, this discrepancy needs to be additionally elucidated.

Previous studies have shown that caspase-8 cleaves Bid. The Mr 15,000 form of truncated Bid binds to Bax and triggers a change in the conformation of Bax. As a result, Bax oligomerizes and inserts into the outer mitochondrial membrane, which results in cytochrome c release from mitochondria (28 , 29) . Cytochrome c in the cytoplasm binds to Apaf-1, which then permits recruitment of procaspase-9. Procaspase-3 is recruited to the Apaf-1/caspase-9 complex and undergoes proteolysis and activation (30) . Interestingly, our studies show that caspase-3 is required for caspase-9 processing (Fig. 2B)Citation . This observation is consistent with the data from Blanc et al. (2) . It is known that procaspase-9 contains a caspase-3 cleavage site, and procaspase-9 is also a substrate of caspase-3 (31) . These results suggest that cross-talk between caspase-3 and caspase-9 (caspase-mediated feedback loop amplification) is essential for the mitochondria-dependent caspase activation.

Caspase-3 is believed to play a pivotal role in apoptotic execution. It promotes apoptotic cell death not only by forming the protease cascades but also by cleaving the substrates in the cells. A growing number of substances cleaved by caspase-3 have been identified, such as PARP (32) , sterol-regulating element-binding protein (33) , gelsolin (34) , the U1-associated Mr 70,000 protein (35) , DNA fragmentation factor (36) , and DNA-dependent protein kinase (17 , 37) . In this study, we observed that the level of Akt protein was reduced by combined treatment with TRAIL and glucose deprivation in MCF-7/casp-3 cells. The reduction of Akt level was due to the cleavage of Akt. Pretreatment with Z-LEHD-FMK and Z-DEVD-FMK, caspase-9, and caspase-3 inhibitor, respectively, inhibited the cleavage of Akt suggesting that inactivation of caspase-3 prevents the cleavage of Akt in MCF-7/casp-3 cells (Fig. 4Citation ; Fig. 5Citation ). Therefore, these results indicate that caspase-3 is involved in the cleavage of Akt, and its cleavage may be one of the facilitating factors during TRAIL-induced apoptotic cell death. Our results are consistent with previous reports that Akt undergoes caspase-mediated cleavage during apoptosis by treatment with Fas ligand, etoposide, or UV irradiation (38) . Akt can directly be cleaved by caspase-3 in vitro (19 , 20) .

Caspases are distinct from other proteases in that they need an Asp-peptide bond in the substrate P1 position to exhibit their proteolytic activities. Previous studies have shown that Akt protein is cleaved by caspases at three sites (TVAD108G, EEMD119F, and SETD434T; Ref. 20 ). The cleavage of Akt protein decreases its kinase activity, which is essential for transmitting the survival signals (20) . Expression of an Akt double mutant at aspartic acid 108 and aspartic acid 119 (D108A+D119A) but not single mutations at either residue is resistant to caspase cleavage and protects cells from apoptotic death (21) . In this study, we observed that Akt was cleaved by caspases at amino acid 108 but not at aspartic acid 119. At the present time, this discrepancy cannot be explained. One possibility is that preferential cleavage sites are dependent on the intracellular level of caspase-3. Aspartic acid 108 may be preferentially cleaved at the relatively low level of caspase-3. In contrast, aspartic acid 119 may be cleaved at the higher level of caspase-3. We believe that this model will provide a framework for future studies.

FOOTNOTES

Grant support: National Cancer Institute Grants CA48000, CA95191, and CA96989; Competitive Medical Research Fund of the University of Pittsburgh Medical Center Health System; Oral Cancer Center Grant Fund; Elsa U. Pardee Foundation; the Pittsburgh Foundation; and Department of Defense Prostate Cancer Research Program PC020530 and FY04 Prostate Cancer Research Program Postdoctoral Traineeship Award.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Yong J. Lee, Department of Surgery, University of Pittsburgh, The Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: (412) 623-3268; Fax: (412) 623-1010; E-mail: leeyj{at}msx.upmc.edu

Received 9/ 1/03; revised 11/25/03; accepted 12/22/03.

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