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[Cancer Research 62, 1433-1438, March 1, 2002]
© 2002 American Association for Cancer Research

Experimental Therapeutics

Extracellular Bad Fused to Toxin Transport Domains Induces Apoptosis

Makoto Ichinose1, Xiu-Huai Liu1, Naoshi Hagihara2 and Richard J. Youle3

Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bad, a proapoptotic member of the Bcl-2 family, is inactivated by phosphorylation, and this loss of activity may contribute to the malignancy of certain types of tumors such as glioblastoma and prostate cancer. To determine whether extracellular Bad can be delivered into cells via cell surface receptor binding and induce apoptosis, we genetically fused the mouse Bad gene to the gene for the translocation and receptor-binding domains of diphtheria toxin (DTTR). The purified Bad (wild-type)-DTTR protein showed cytotoxicity to human glioma cells in a dose-dependent manner. Bad phosphorylation sites at codons 112 and 136 were mutated from serine to alanine to prevent Bad inactivation by kinases and to increase the toxicity of Bad. The Bad (S112A S136A)-DTTR protein was at least 5 times more toxic than Bad (wild-type)-DTTR with an IC50 of 5 x 10-8 M. The Bad (S112A S136A)-DTTR protein altered the subcellular distribution of Bcl-XL, indicating that it enters the cell cytoplasm and binds Bcl-XL. Bad (S112D S136A)-DTTR, mutated to mimic phosphorylation of Bad, showed lower toxicity than either Bad (wild-type)-DTTR or Bad (S112A S136A)-DTTR, additionally indicating that Bad-DTTR must bind Bcl-XL to stimulate apoptosis. We conclude that extracellular Bad can be delivered into cells via the transport domain of a bacterial toxin and may be used to induce apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteins in the Bcl-2 family are key regulators of apoptosis that may either promote cell death or inhibit cell death (1 , 2) . Heterodimerizaton between prosurvival Bcl-2 family members and proapoptotic family members is considered to regulate apoptosis (3 , 4) . Bad, a proapoptotic member of the Bcl-2 family, binds and antagonizes prosurvival Bcl-XL and induces apoptosis (4) . Bad protein contains 23 serines and 10 threonines within 204 amino acids, and among of them, serine 112, 136, and 155 have been identified as phosphorylation sites (5, 6, 7, 8, 9, 10, 11, 12, 13) . Phosphorylation of these serines inhibits binding of Bad to prosurvival protein Bcl-XL on the outer membrane of mitochondria, and Bad changes subcellular distribution from the mitochondria to bind the 14-3-3 protein in the cytosol (5, 6, 7, 8, 9, 10, 11, 12, 13) . Thus, Bad regulates apoptosis from the cell cytoplasm and must be expressed in or delivered into the cytosol to be used as a death promoter. One approach to deliver such an exogenous protein to the cytosol is through the use of genetic vectors. However, when this approach is used for the treatment of diseases such as malignant tumors, the potential pathogenicity of the vectors poses significant risk for the patient.

Alternatively, bacterial toxins such as DT,4 pseudomonas exotoxin, and anthrax toxin that reach the cytosol by translocation through endosomal membranes (14, 15, 16, 17) have been used to deliver peptides or whole proteins into cells (18, 19, 20) . Delivery of antigenic peptides such as toxin fusion proteins was efficient in stimulating the MHC class I immunity pathways (19 , 20) .

DT consists of three structurally and functionally distinct domains: (a) the A chain that ADP-ribosylates elongation factor 2 and inactivates translation of protein synthesis; (b) a cell-surface receptor-binding domain; and (c) a translocation domain that facilitates transport of the A chain into the cytosol (21) . We showed previously that Bcl-XL fused to DT (22) or anthrax toxin (23) domains could be delivered into cells to inhibit apoptosis in vitro and in vivo. In the current study, we fused mouse Bad to the DTTR. The fusion protein was effective at inducing apoptosis in human glioma cells and prostate cancer cells. The mutant Bad proteins, which have substitutions of serine to alanine at two or three phosphorylation sites, showed more potent ability to induce apoptosis than wild-type Bad. We demonstrated that extracellular Bad could be delivered into cells via the transport domain of a bacterial toxin and induced apoptosis in tumor cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
U251, U87, and U138 human glioma cells; PC-3, DU145, and TSU human prostate cancer cells; 9 L rat gliosarcoma; C6 rat glioma cells; and Cos-7 green monkey renal epithelial cell lines (American Type Culture Collection, Rockville, MD) were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) containing 2 mM of glutamine, 1 x nonessential amino acids, 2.5 mM of sodium pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin (all from Biofluids Inc., Rockville, MD), 50 µg/ml gentamicin (Life Technologies, Inc.), and 10% heat-treated FCS. All of the cells were cultured in T75 flasks at 37°C in 5% CO2.

Construction of the Bad-DTTR Expression Plasmid.
Substitution of serine to alanine (TCG->GCG) or aspartate (TCG->GAC) at serine positions 112, 136, and 155 of Bad was performed by PCR-based site-directed mutagenesis. The full-length mouse Bad gene (codon 1–204) with or without serine mutations and the DT gene from codons 194 through 535 (DTTR) were amplified by PCR. For the fused gene, the reverse primer of Bad and the forward primer of DTTR contained ends of common sequence. These two PCR products, Bad (wild-type or mutant) and DTTR, were used as templates to directly fuse the Bad gene to the 5' end of the DTTR gene by second-round PCR with primers including NdeI and XhoI restriction site. The Bad (wild-type or mutant)-DTTR gene fragment digested with NdeI and XhoI was ligated into the prokaryotic expression vector pET16b (Novagen, Inc., Madison, WI) cut with NdeI and XhoI. The full-length Bad (codons 1–204) and DTTR (codons 194–535) genes were separately subcloned into pET16b vectors through NdeI and XhoI sites for the controls. The histidine tag and Factor Xa digestion site sequences from the expression vector were upstream of these gene coding sequences. The expression gene constructions were verified by DNA sequencing.

Purification and SDS-PAGE Characterization of Bad-DTTR.
Escherichia coli BL21(DE3; Novagen) was used to express wild-type or mutated Bad-DTTR, Bad (S112A S136A), and DTTR. Recombinant bacteria were grown in 1 liter of LB medium (Digene, Beltsville, MD) containing 50 µg/ml ampicillin (Sigma Chemical Co., St. Louis, MO) in 2-liter flasks at 37°C. Protein expression was induced by addition of 1 mM of isopropyl-ß-D-thiogalactopyranoside (Life Technologies, Inc.) when the OD600 reached 0.5–0.7. After a 2.5-h incubation, cells were harvested by centrifugation at 5,000 x g, and pellets were lysed by French press. The inclusion bodies were collected by centrifugation at 20,000 x g and dissolved in 6 M guanidine-HCl. His-Bind Resin (Novagen, Inc.) was used to purify the proteins. The proteins were refolded by rapid dilution in a 100-fold volume of the refolding buffer [100 mM Tris acetate (pH 8.0) and 0.5 M arginine] followed by incubation at 4°C for >=24 h. The refolded proteins were concentrated with polyethylene glycol 15,000–20,000 and dialyzed against PBS. The proteins were subjected to 10–20% SDS-PAGE and stained with Brilliant Blue R (Sigma).

Cytotoxic Assay.
To access the cytotoxicity of the recombinant proteins, three kind of assays were performed: cellular protein synthesis inhibition, apoptotic cell counting, and clonogenic cell viability assays. Cellular protein synthesis inhibition was determined as described previously (24 , 25) with slight modifications. Briefly, cells in 100 µl were incubated at concentrations of 1 x 105 cells/ml in 96-well microtiter plates overnight and treated with various concentrations of purified proteins for 72 h in leucine-free RPMI 1640 followed by a 1-h pulse with 0.1 mCi [14C]leucine. Then, cells were harvested onto glass fiber filters using a PHD cell harvester (Cambridge Technology, Watertown, MA), and the radioactivity was counted using a liquid scintillation counter (Wallac Oy, Turku, Finland). The results were expressed as a percentage of radiolabeled leucine incorporation in PBS-treated control cells. Values given represent the average of triplicate samples with <10% SD.

For counting apoptotic cells, cells were seeded on 96-well plates as described above. The cells were treated for various time periods, and apoptotic cells were quantified by staining with Hoechst 33342 dye (Sigma). At least three fields were counted in each of at least three wells using a model IX70 confocal microscope (Olympus America Inc., Eugene, OR) with the 364-nm line of an argon laser for excitation of Hoechst 33342 dye. Results were calculated as the percentage of apoptotic cells/total cells/defined field.

The clonogenic cell viability assay was performed as described previously (26) with slight modifications. Briefly, U251 cells in 2 ml at concentrations of 2 x 102 cells/ml were incubated in six-well plates in triplicate and incubated overnight to allow cells to attach. The cells were exposed to PBS or various Bad-DTTR fusion proteins at 1.31 x 10-6 M for 14 days. After the cells were washed and stained with crystal violet (0.25% in 25% ethanol), six-well plates were directly scanned by an image scanner with 300-dpi resolution. The area of stained colonies was measured with NIH Image software.

Subcellular Localization of GFP-Bcl-XL ({Delta} leucine 229) in COS-7 Cells.
According to the manufacturer (Boehringer Mannheim Co., Indianapolis, IN), 16 h before transfection, 1 x 105 Cos-7 cells in 2 ml were seeded in 35-mm dishes. FuGENE 6 (5 µl) was diluted in 100 µl of OPTI-MEM I (Life Technologies, Inc.). The FuGENE 6 dilutions were kept for 5 min at room temperature before adding 1 µg of pEGFP-Bcl-XL ({Delta} leucine 229). The DNA/FuGENE 6 mixture was mixed by gently tapping the tube and left at room temperature for 15 min for complexing. Thereafter, 100-µl drops were transferred to Cos-7 cells cultured in 35-mm dishes. Three hours after transfection, cells were incubated with 2 x 10-5 M zBoc-fmk (caspase inhibitor) for 1 h to delay apoptosis and treated with PBS plus 1 x 10-7 M STS or 1.31 x 10-6 M Bad (S112A S136A)-DTTR plus 1 x 10-7 M STS. After cells were incubated for 30 min, 20 ng/ml of a mitochondrion-specific dye (Mitotracker Red CMXRos; Molecular Probes Inc., Eugene, OR) was added. Another 30 min later, images were collected on the confocal microscope above. The 488- and 568-nm lines of krypton/argon laser were used for fluorescence excitation of GFP and mitotracker red CMXRos, respectively.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Purification of Bad-DTTR Fusion Protein.
To deliver extracellular Bad protein into cells via cell surface receptor binding, we genetically fused the full-length mouse Bad gene to the DTTR (Fig. 1A)Citation . The fusion protein was expressed in E. coli with the pET16b vector, purified with histidine-binding resin, and refolded by rapid dilution. SDS-PAGE showed that the purified Bad-DTTR protein migrated at approximately Mr 62,000 that corresponds to the expected size (Fig. 1B)Citation . The final protein preparation was >95% homogeneous.



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  		Fig. 1. Construction and SDS-PAGE of Bad-DTTR. A, schematic diagram of the chimera, Bad-DTTR. The fused gene, Bad (wild-type or mutant)-DTTR, was cloned into the vector, pET16b, yielding a histidine tag sequence at the NH2 terminus of the fused gene. B, SDS-PAGE of purified DTTR, Bad (S112A S136A), Bad (S112A S136A)-DTTR, and Bad (wild-type)-DTTR proteins. Purified proteins were subjected to SDS-PAGE (10–20%) and visualized by coomasie brilliant blue staining. Lane A, DTTR; Lane B, Bad (S112A S136A); Lane C, Bad (S112A S136A)-DTTR; Lane D, Bad (wild-type)-DTTR. C, toxicity of Bad-DTTR to U251 human glioma cells. Incorporation of radiolabeled leucine by U251 cells after 72-h treatment with the Bad (wild-type or mutant)-DTTR protein was measured and presented as a percentage relative to PBS-treated cells. The mean values determined from triplicate measurements are plotted versus concentration of the fusion protein. {square}, Bad (wild-type)-DTTR; {blacksquare}, Bad (S112A S136A)-DTTR.

 
Bad Fused with Two DT Domains Induces Apoptosis.
To determine the bioactivity of the Bad-DTTR fusion protein, U251 glioma cells were incubated with fusion protein for 72 h, and the cytotoxicity was measured by [14C]leucine incorporation assay. The cytotoxicity of Bad-DTTR was seen in a dose-dependent manner; however, it did not show enough activity to determine an IC50 up to 10-7 M concentrations (Fig. 1C)Citation . To increase the cytotoxicity of Bad, we mutated Bad from serine to alanine at codons 112 and 136 (S112A S136A) to prevent Bad inactivation by phosphorylation at these serines (5, 6, 7, 8) . The mutant Bad (S112A S136A) was more toxic than Bad (wild-type) after transfection into 9 L cells (data not shown), consistent with previous reports (7 , 8) . We produced the mutated Bad (S112A S136A) protein fused to DTTR by the same methodology with which Bad (wild-type)-DTTR protein was produced (Fig. 1B)Citation . The Bad (S112A S136A)-DTTR protein showed a dose-dependent toxicity with an IC50 ~5 x 10-8 M (Fig. 1C)Citation , at least five times more potent than Bad (wild-type)-DTTR (Fig. 1C)Citation . Neither Bad (S112A S136A) nor DTTR proteins alone were toxic to U251 cells (Fig. 2A)Citation . The potential for Bad-DTTR to induce apoptosis was measured directly by quantitating the number of cells undergoing apoptosis as visualized by nuclear condensation using the Hoechst dye, 33342. After a 48-h exposure of U251 glioma cells to Bad (S112A S136A), >25% of the cells were in the process of apoptosis. In contrast, the DTTR domain alone or Bad (S112A S136A) alone caused <3% apoptosis (Fig. 2B)Citation . Bad (S112A S136A)-DTTR showed a time-dependent induction of apoptosis (Fig. 2C)Citation . To examine the role of the DT receptor binding in the toxicity of Bad (S112A S136A) DTTR, an excess of DTTR was incubated with cells during exposure to Bad (S112A S136A) DTTR. Table 1Citation shows that excess DTTR competed very effectively for Bad (S112A S136A) DTTR toxicity. To confirm the role of DT receptor binding in cytotoxicity of Bad-DTTR, mouse cells lacking the DT receptor (27) were compared with human cells expressing the receptor. Human cell lines including glioma cell lines (U87, U138, and U251) and prostate carcinoma cell lines (PC3, DU145, and TSU) were sensitive to apoptosis induced by Bad (S112A S136A)-DTTR (27–45%), whereas the rat cell lines 9 L gliosarcoma and C6 glioma were relatively resistant to apoptosis induced by this fusion protein (9–15%; Fig. 3Citation ). These results indicate that the fusion protein is delivered to cells via the DT receptor and induces apoptosis.



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  		Fig. 2. Toxicity of Bad (S112A S136A)-DTTR to U251 human glioma cells. A, cellular protein synthesis inhibition by Bad (S112A S136A)-DTTR. U251 cells were treated with various concentrations of the indicated proteins or PBS for 72 h. Cellular protein synthesis inhibition was measured and expressed as described in the legend of Fig. 1CCitation . {circ}, Bad (S112A S136A); {triangleup}, DTTR; {blacksquare}, Bad (S112A S136A)-DTTR. B, apoptosis is induced by Bad (S112A S136A)-DTTR. U251 cells were incubated with PBS or 1.31 x 10-6 M of the indicated proteins for 48 h. Apoptosis was measured by quantitating chromatin condensation using Hoechst 33342. C, apoptosis induced by Bad (S112A S136A)-DTTR. U251 cells were incubated with PBS ({diamond}) or 1.31 x 10-6 M Bad (S112A S136A)-DTTR ({blacksquare}) for 4–48 h. Apoptotic cells stained with Hoechst 33342 dye were counted and expressed as a percentage of total cells at the indicated time points. The data shown are the mean of triplicate values for each time point; bars, ±SD.

 

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  		Table 1 Competition of Bad-DTTR toxicity by excess DTTR domain

 


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  		Fig. 3. Apoptosis of various cell lines induced by Bad (S112A S136A)-DTTR. The indicated cell lines were incubated with 1.31 x 10-6 M Bad (S112A S136A)-DTTR for 48 h. Apoptotic cells stained with Hoechst 33342 dye were counted and expressed as a percentage of total cells. The data shown are the mean of triplicates; bars, ±SD.

 
Entry of the Bad Fusion Protein into the Cytosol Compartment.
Although the fusion protein was delivered to cells via the DT receptor, it remains unclear whether Bad enters the cell cytosol to induce apoptosis. During apoptosis, the heterodimerization of Bad and Bcl-XL appears to be an important mechanism for the proapoptotic function of Bad (8 , 28) . To test whether the fusion protein can enter cells and associate with Bcl-XL, we examined the effect of the fusion protein on the subcellular distribution of Bcl-XL in the cell. Cos-7 cells were transfected with GFP-Bcl-XL ({Delta} leucine 229) containing a mutation in the membrane anchor domain, which causes Bcl-XL to locate diffusely in the cell cytoplasm even after cells are exposed to apoptotic stimuli (Fig. 4, a and b)Citation . Cotransfection of Bad with the GFP-Bcl-XL ({Delta} leucine 229) causes Bcl-XL ({Delta} leucine 229) to translocate to the mitochondrita.5 When cells are treated with the Bad (S112A S136A)-DTTR fusion protein and 1 x 10-7 M STS, the distribution of GFP-Bcl-XL ({Delta} leucine 229) changes to a punctate mitochondrial distribution (Fig. 4, c and d)Citation . Hence, Bad-DTTR fusion protein appears to enter cells and associate with Bcl-XL in the cytoplasm changing the distribution of Bcl-XL from the cytoplasm to the mitochondria. This binding may inactivate Bcl-XL and mediate the proapoptotic activity of Bad-DTTR.



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  		Fig. 4. Effect of Bad (S112A S136A)-DTTR on the distribution of Bcl-XL. Cos-7 cells transfected with GFP-Bcl-XL ({Delta} leucine 229) were treated with PBS (a and b) or with Bad (S112A S136A)-DTTR protein (c and d) and a low concentration of STS (1 x 10-7 M; a–d). Confocal images were taken with the appropriate wavelength for GFP (a and c) to follow Bcl-XL distribution and for Mitotracker Red CMXRos (b and d) to visualize mitochondria independently.

 
Bioactivity of Bad-DTTR Correlates with Bcl-XL Binding.
It is known that dephosphorylated Bad interacts with and antagonizes the prosurvival activity of Bcl-XL; however, it is still unclear whether the apoptosis-inducing ability of Bad occurs by this mechanism. To determine whether the Bad-DTTR fusion protein induces apoptosis by association with Bcl-XL, we constructed Bad with a serine to asparate mutation at codon 112, which mimics phosphorylation and prevents binding to Bcl-XL (29) . The cytotoxicity of Bad (S112D S136A)-DTTR was less than that of either Bad (S112A S136A)-DTTR or Bad (wild-type)-DTTR (Fig. 5)Citation . This indicates that the association with Bcl-XL contributes to the proapoptotic activity of this fusion protein. Because inhibition of protein synthesis can reflect transient nonlethal effects, we also performed clonogenic assays of the three Bad-DTTR fusion proteins. U251 cell colonies (Fig. 6A)Citation in six-well plates after incubation with fusion proteins for 2 weeks were quantitated (Fig. 6B)Citation . Both the size and number of U251 colonies was reduced by the fusion proteins. The total colony area analysis showed that Bad (S112D S136A)-DTTR was less toxic than Bad (wild-type)-DTTR and Bad (S112A S136A)-DTTR (Fig. 6B)Citation . Bad (S112A S136A)-DTTR killed >90% of the cells effecting 1 log kill at 1.31 x 10-6 M.



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  		Fig. 5. Comparing the toxicity of Bad (S112D S136A)-DTTR to that of Bad (S112A S136A)-DTTR. U251 cells were treated with various concentrations of the indicated proteins or PBS. Cellular protein synthesis inhibition was measured and expressed as described in the legend of Fig. 1CCitation . {blacksquare}, Bad (S112A S136A)-DTTR; {diamond}, Bad (S112D S136A)-DTTR; {square}, Bad (wild-type)-DTTR; bars, ±SD.

 


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  		Fig. 6. Inhibition of U251 cell growth and viability by the fusion proteins. Cells were plated sparsely, treated with various Bad-DTTR fusion proteins at 1.31 x 10-6 M, and incubated for 14 days. A, scanned images of U251 colonies treated with PBS (a), Bad (wild-type)-DTTR (b), Bad (S112A S136A)-DTTR (c), and Bad (S112D S136A)-DTTR (d) are shown after staining with crystal violet. B, the areas of colonies were measured by NIH Image software and presented as pixels. The data shown are the mean of triplicate; bars, ±SD.

 
Recently, a novel phosphorylation site was discovered on serine 155 within the Bad BH3 domain (9, 10, 11, 12, 13) that blocks the ability of Bad to interact with Bcl-XL. To test the importance of the phosphorylation status of serine 155 on the bioactivity of the Bad fusion protein, we produced a triple mutant Bad, Bad (S112A S136A S155A)-DTTR, and performed protein synthesis and clonogenic bioassays. Bad (S112A S136A S155A)-DTTR showed no greater inhibition of U251 cell growth than Bad (S112A S136A)-DTTR (Fig. 7Citation ; data not shown). This indicates that serine 112 and serine 136 regulate serine 155 phosphorylation consistent with the recent model by Datta et al. (10) .



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  		Fig. 7. Comparing the toxicity of Bad (S112A S136A S136A)-DTTR to that of Bad (S112A S136A)-DTTR. U251 cells were treated with various concentrations of indicated proteins or PBS. Cellular protein synthesis inhibition was measured and expressed as described in the legend of Fig. 1CCitation . {blacksquare}, Bad (S112A S136A)-DTTR; {diamond}, Bad (S112A S136A S155A)-DTTR; bars, ±SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Delivery of peptides and proteins through the plasma membrane could greatly enhance translation of basic research discoveries into therapeutics. One strategy of transporting peptides into cells has been to use bacterial toxins such as DT, pseudomonas exotoxin A, and anthrax toxin (30) as carriers. Common features of bacterial toxins include binding to the cell surface, internalization into an endosome, translocation to the cytosol, and then catalytic inactivation of cellular proteins, leading to cell death (31) . The catalytic subunit of bacterial toxins can be exchanged with other polypeptides to introduce exogenous peptides and proteins into the cytosol without toxicity (18 , 20 , 32) . With this approach, we showed previously that the prosurvival Bcl-2 family member Bcl-XL fused either to the receptor-binding domain of DT (22) or to the lethal factor of anthrax toxin (23) could be delivered into cells and prevent apoptosis in vitro and in vivo. Although the bacterial transport system was not used, Bax fused to interleukin 2 was found also to specifically target interleukin 2 receptor-expressing cells and induced cell-specific apoptosis (33) . Accordingly, both prosurvival and proapoptotic Bcl-2 family members have been introduced into cells as extracellular proteins and showed apoptosis-regulating bioactivity.

In this study, we asked if the proapoptotic Bcl-2 family member Bad could be delivered to cells to induce apoptosis, because Bad functions downstream of the tumor suppressor PTEN and may display anticancer activity physiologically. The Bad (wild-type)-DTTR protein showed dose-dependent toxicity to U251 cells. Akt and other kinases inactivate Bad by phosphorylation of serines, suggesting that Bad delivered to cells via the DTTR could be inactivated by phosphorylation. Thus, we mutated Bad at serine 112 and 136, and found the mutant was more toxic than Bad (wild-type). This is compatible with the model that only nonphosphorylated Bad can associate with Bcl-XL and induce apoptosis (34) , and indicates that the Bad fusion protein is entering the cell and effecting cell death by association with Bcl-XL. During apoptosis, Bad (S112AS136A)-DTTR changed the distribution of Bcl-XL from the cytosol to the mitochondria (Fig. 4)Citation , additionally indicating that this fusion protein can enter the cytosol and associate with Bcl-XL.

Bad is phosphorylated at serine 112 by the ribosomal S6 kinases and a mitochondria-anchored cyclic AMP-dependent protein kinase A (5 , 6) and at serine 136 by Akt kinase (7 , 8) . In addition to these two phosphorylation sites, it has been reported recently that Bad also can be phosphorylated at serine 155, in the middle of the BH3 domain, by protein kinase A, and this induces complete dissociation of Bad from Bcl-XL (9, 10, 11, 12, 13) . We compared the toxicity of Bad (S112A S136A S155A) with that of Bad (S112A S136A) both fused to DTTR. The triple-mutant Bad showed no more toxicity than the double-mutant Bad. This is consistent with the sequential phosphorylation model for Bad inactivation (10) where serine 136 phosphorylation is required for serine 155 phosphorylation.

The Akt kinase, a growth factor-regulated serine/threonine kinase activated by the phosphatidylinositol 3'-kinase signaling pathway (35 , 36) , is deregulated in a variety of tumors by several different mechanisms: by extracellular survival factors, by activating mutations in the Ras oncogene, or by a deletion/inactivating mutation of the PTEN/MMAC1 gene (37, 38, 39) . The PTEN/MMAC1 tumor suppressor gene, was mapped to 10q23 (40) . Homozygous deletions of PTEN/MMAC1 occur in 7%, and mutations of a single retained PTEN/MMAC1 allele occur in 40% of glioblastomas (41) . Advanced prostate cancers also show frequent loss of PTEN/MMAC1 expression (40 , 42, 43, 44, 45) . Davies et al. (46 , 47) showed PTEN/MMAC1 transfection of U251 cells with an adenovirus vector-inhibited Akt-mediated signaling, phosphorylation of Bad, and cell growth. They also showed this transfection induced apoptosis in prostate cancer cell line LNCaP via Akt/PKB activation. These results imply that loss of PTEN increases cell proliferation and contributes to the malignancy. U251 and U87 glioma cells, and PC3 prostate cancer cells used in our study either fail to express PTEN or have PTEN mutations (48 , 49) . These cell lines all show sensitivity to apoptosis induced by the Bad (S112A S136A)-DTTR fusion protein, which cannot be inactivated by Akt phosphorylation. This indicates that unphosphorylated Bad delivered extracellularly is available to induce apoptosis in tumor cells with mutated PTEN/MMAC1 such as glioblastomas and prostate cancers. In the future, although we have to consider specificity for this delivery system, introduction of proapoptotic proteins such as Bad into cells might be a new approach for the treatment of tumors. Linkage of the Bad fusion protein to cell type-specific ligands or antibodies may allow tumor-specific targeting of Bad as has been accomplished with DT and other protein toxins (50 , 51) .


   ACKNOWLEDGMENTS
 
We thank Dr. Motoshi Suzuki for valuable discussions and technical assistance.


   FOOTNOTES
 
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.

1 These authors contributed equally to this study. Back

2 Present address: Department of Neurosurgery, Saga Medical School, 5-1-1 Nabeshima, Saga, 849-8501 Japan. Back

3 To whom requests for reprints should be addressed, at Biochemistry Section, Building 10, Room 5D-37, MSC 1414, 10 Center Drive, Bethesda, MD 20892-1414. Phone: (301) 496-6628; Fax: (301) 402-0380; E-mail: youler{at}ninds.nih.gov. Back

4 The abbreviations used are: DT, diphtheria toxin; DTTR, translocation and receptor-binding domains of DT; STS, staurosporine; PTEN, phosphatase and tensin homologue deleted on chromosome ten; GFP, green fluorescent protein. Back

5 K. Sanders and R. J. Youle, unpublished observations. Back

Received 7/ 2/01. Accepted 12/21/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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