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Vol. 1, 95-102, December 2001     Molecular Cancer Therapeutics
© 2001 American Association for Cancer Research

The Pro-apoptotic Protein, Bik, Exhibits Potent Antitumor Activity That Is Dependent on Its BH3 Domain1

Yiai Tong, Quan Yang, Carol Vater, L.K. Venkatesh, Dominique Custeau, Thomas Chittenden, G. Chinnadurai and Henriette Gourdeau2

Shire BioChem, Inc., Laval, Québec, H7V 4A7 Canada [Y. T., Q. Y., D. C., H. G.]; The Institute for Molecular Virology, St. Louis University Medical Center, St. Louis, Missouri 63110 [L. K. V., G. C.]; and Apoptosis Technology, Inc., Cambridge, Massachusetts 02139 [C. V., T. C.]


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Bcl-2 homology 3 (BH3) domain is present in most members of the Bcl-2 protein family and is required to confer the death-inducing properties of pro-apoptotic members, including Bax, Bak, Bad, and Bik, in cell-based assay systems. To determine whether the BH3 domain possesses a similar role in tumor tissues in vivo, we overexpressed the wild-type Bik protein and its BH3-deleted counterpart, using adenoviral technology, in chemoresistant human tumor prostate (PC-3) and colon (HT-29) cell lines growing in vitro and in vivo. Bik caused apoptosis in both PC-3 and HT-29 cells in vitro by inducing the release of cytochrome c from mitochondria to cytoplasm, resulting in the catalytic activation of caspases 9, 7, and 3 and cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. When the BH3 domain was deleted from the Bik protein, no effect on mitochondrial activity or cell morphology could be observed. Furthermore, intratumoral injection of an adenovirus vector expressing the Bik gene, but not the deleted BH3 Bik gene, suppressed the growth of PC-3 and HT-29 xenografts established in nude mice. Histological examination of tumors from mice treated with the wild-type Bik adenoviral construct demonstrated cellular debris, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive staining, and morphological changes associated with apoptosis. In contrast, tissue sections obtained from tumors treated with the BH3-deleted Bik adenoviral construct showed no evidence of apoptosis. Thus, our results suggest that the BH3 domain is required for the antitumor activity of the Bik protein and provides a novel therapeutic approach for cancer therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is a physiological process by which cells activate their own destruction to maintain tissue homeostasis during embryonic development. Deregulation of this process contributes to a number of human diseases that are caused either by an increase in cell death, such as neurodegenerative disorders and AIDS, or enhanced cell survival, such as cancer and autoimmune diseases (1). It is thought that cancers are inherently resistant to current chemotherapy because they fail to respond normally to apoptosis signals (2, 3). The Bcl-2 family of proteins are key regulators of apoptosis and play an essential role in cancer and chemoresistance (4). The Bcl-2 family is composed of two groups of proteins having antagonist functions: the pro-apoptotic members, such as Bax, Bik, Bak, and Bad, and the anti-apoptotic members, such as Bcl-2 and Bcl-XL. These proteins contain conserved BH3 domains important for homo or hetero-dimerization (5, 6). The BH3 domain of the pro-apoptotic members is required for these proteins to hetero-dimerize with their antiapoptotic counterpart to induce apoptosis (7-9). Indeed, recent studies have indicated that synthetic BH3 peptides could induce a caspase-dependent apoptotic response in vitro (10, 11) and increase survival of human myeloid leukemia-bearing mice (11).

Bik is a pro-apoptotic member (12) and shares only the BH3 domain with Bcl-2 family of proteins and, hence, is designated as a "BH3-only" protein. Recent studies have shown that overexpression of the Bax gene (13, 14) or the Bak gene (15) in human tumor xenografts led to potent in vivo antitumor activity. In this study, we wanted to determine whether the Bik gene had similar antitumor activity. Our results indicated that Ad-mediated overexpression of Bik induces programmed cell death of tumor cells by triggering the mitochondrial pathway of apoptosis both in vitro and in vivo. Furthermore, the potent antitumor activity of Bik was dependent on its BH3 domain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
The prostate (PC-3) and colorectal adenocarcinoma (HT-29) human tumor cell lines were obtained from the American Type Culture Collection (Manassas, VA) and maintained in Ham’s F-12 and McCoy’s 5A, respectively, supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 µ/ml streptomycin (Life Technologies, Inc., Burlington, Ontario, Canada).

Construction and Production of Bik Ads
DNA fragments containing the cytomegalovirus promoter and HA-tagged version of Bik cDNA (12) or the BH3 deletion ({Delta}BH3) mutant (7) were derived from pcDNA3-based expression vectors. These DNA fragments were subcloned into an Ad gene transfer vector that contains the Ad5 sequences spanning nucleotides 1–5778 to generate Ad-wtBik and Ad-{Delta}Bik ({Delta}BH3), respectively. In Ad-wtBik and Ad-{Delta}Bik, the Bik transcription cassettes (under the control of the cytomegalovirus promoter and interleukin-2 poly(A)+ signal) are located between nucleotide positions 3322 and 451 (in right-to-left transcriptional orientation). To produce recombinant Ads, 293 cells were transfected with 5 µg of Ad-wtBik or Ad-{Delta}Bik DNA and 1 µg of Ad5 (wt) DNA, exhaustively digested with restriction endonuclease ClaI. Transfected cells were overlaid with 0.8% agarose in DMEM (Life Technologies, Inc.) supplemented with 2.5% heat-inactivated fetal bovine serum. Viral plaques were visualized by staining with neutral red, isolated, and amplified. In certain instances, the recombinant virus expressing wt Bik was also amplified in a 293 cell line that ectopically overexpresses the antiapoptotic protein Bcl-XL. The recombinant Ads were positively identified on the basis of detailed restriction analysis of viral DNA. The viral isolates exhibiting the expected restriction patterns were further analyzed for expression of HA epitope-tagged Bik by immunoprecipitation analysis as described previously (16). Amplification of the Bik Ads and determination of pfu were performed by Quantum Technologies, Inc. (Montréal, Québec, Canada).

Cytotoxicity and Apoptosis Assays
PC-3 and HT-29 cells were plated in their respective medium in 96-well tissue culture plates at 2 x 103cells/well, 24 h before infection. Cells were infected by adding control Ad (Ad-{Delta}Bik) or Ad-wtBik directly to the cell culture media at final concentrations ranging from 0 to 640 pfu/cell. After a 48-h infection with the Ads, impaired cell viability was determined by adding MTT, which measures mitochondrial oxidoreductase activity, at a final concentration of 0.4 mg/ml. After a 4-h incubation, the resulting purple formazan precipitate was solubilized by adding 150 µl of DMSO, and absorbance was quantified at 590 nm with a Dynatech MR5000 plate reader. Morphological evaluation of cells for apoptosis was done by Hoechst 33343 (Molecular Probes, Inc., Eugene, OR) staining of paraformaldehyde-fixed cells after a 24-h incubation with 250 pfu/cell Ad-{Delta}Bik (control) or Ad-wtBik. Fluorescence was observed on a Leica-inverted microscope equipped with a fluorescence module.

DNA Fragmentation
PC-3 and HT-29 cells were harvested 48 h postinfection with 250 pfu/cell Ad-{Delta}Bik (control) or Ad-wtBik. Cells were trypsinized, washed in cold PBS buffer, and centrifuged, and the cell pellet was resuspended in 400 µl of lysis buffer [5 mM Tris-HCl (pH 7.5), 5 mM EDTA, and 0.5% Triton X-100]. The cell suspension was centrifuged to remove cell debris, and the supernatant was precipitated with ethanol. The pellet, containing genomic DNA, was resuspended in proteinase K buffer containing 100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 0.2% SDS, and 200 mM NaCl and incubated with 10 µg/ml RNase A for 30 min at 37°C, followed by digestion with 100 µg/ml proteinase K at 37°C for 3 h. The DNA fragments were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining.

Western Blot Analysis
Sub-confluent cultures of PC-3 and HT-29 cells were treated with 250 pfu/cell Ad-{Delta}Bik (control) or 250 pfu/cell Ad-wtBik for 24 and 48 h. Cells were then harvested by trypsinization and recovered by centrifugation (500 x g, 10 min), and the resulting cell pellets were processed for cytochrome c extraction or caspase activity determination as described below. For cytochrome c analysis, the cell pellets were resuspended in cold mitochondrial buffer [10 mM HEPES (pH 7.0), 210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin A, and 10 µg/ml leupeptin; Ref. 17], lysed by passing six times through a 25-gauge needle, and centrifuged for 3 min at 4°C at 10,000 x g. The supernatant, containing the released cytochrome c, was collected, mixed with an equal volume of 2 x SDS protein loading buffer, and separated on SDS-polyacrylamide gel under reducing conditions. Western blot analysis was done using a mouse monoclonal antihuman cytochrome c antibody (lot #BR101; R&D Systems, Inc., Minneapolis, MN), followed by enhanced chemiluminescence detection (Amersham Pharmacia Biotech) as described previously (18). For caspase and PARP analysis, cell pellets were lysed directly in 200 µl of SDS protein loading buffer separated as described above. The individual caspases and PARP content were detected using the apoptosis sampler kit (#9915; Cell Signaling Technology and New England BioLabs, Inc., Mississauga, Ontario, Canada). Actin antibody (C-11; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used as protein loading control. To verify exogenous Bik expression, the blots were probed with the HA-11 mouse monoclonal antibody (Babco Berkeley antibody company, Berkeley, CA). For reprobing, membranes were stripped by treating with 62.6 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM ß-mercaptoethanol for 20 min at 50°C and then washed five times in Tris-buffered saline containing 5% Triton X-100 buffer at room temperature.

Animal Studies
CD-1 male or female nude mice (6–8 weeks old; Charles River, Québec City, Canada) were injected s.c. with 2 x 106 PC-3 or HT-29 cells, respectively. Nude mice were maintained in a P2 level facility using a previously approved animal care committee protocol. Tumor measurements, taken by calipers in two dimensions twice weekly, were converted to tumor volumes using the formula, volume (mm3) = width (mm2) x length (mm)/2. Animals bearing tumors were randomized (four to seven per group) before treatment. Administration of the Ads (intratumoral injections) began once tumors reached an average volume of 40 mm3 (PC-3 xenograft) or 80 mm3 (HT-29 xenograft). For PC-3 xenograft tumor model, Ad-{Delta}Bik (control) or Ad-wtBik were injected intratumorally at 2 x 109 pfu or 20 x 109 pfu twice (48 h apart). For HT-29 xenograft tumor, mice received four intratumoral injections (48 h apart) of Ad-{Delta}Bik (control) or Ad-wtBik of 15 x 109 pfu/dose. The experiment was terminated when average tumor volumes in the control saline-treated group reached a median tumor volume of 150 or 600 mm3 for PC-3 and HT-29 xenografts, respectively.

All animals received humane care in compliance with the Canadian Council Guidelines to the care and use of experimental animals. These in vivo experimental protocols were approved by Shire BioChem’s animal care committee, and our animal facility is accredited by the Canadian Council for animal care.

Histochemical Study
Two mice per tumor model received two consecutive intratumoral injections of either Ad-{Delta}Bik (control) or Ad-wtBik. Tumors were excised, fixed in 4% paraformaldehyde overnight, embedded in Cryomatrix, and sectioned with a Leica cryostat at a thickness of 6 µm. Tissue sections were either stained for H&E (overall morphological observation) or labeled with the TUNEL reaction mixture (detection of apoptosis) as described previously (19).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bik Overexpression Is Toxic and Dependent on Its BH3 Domain
The MTT assay was used to determine whether overexpression of Bik had any effect on the mitochondrial activity of PC-3 and HT-29 cell lines. Logarithmically growing cells were infected either with Ad-wtBik or Ad-{Delta}Bik for 48 h. Whereas Ad-wtBik was quite toxic to cells, causing mitochondrial toxicity at concentrations as low as 20 pfu/cell, the Ad-{Delta}Bik, where the BH3 domain has been deleted, had no effect on mitochondrial activity, even at doses of >=640 pfu/cell (Fig. 1). HT-29 cells were more sensitive to Ad-wtBik mitochondrial toxicity compared with PC-3 cells, where a 50% loss of mitochondrial activity was observed with 80 pfu/cell compared with 320 pfu/cell, respectively. Exogenous expression of Bik was found to be similar in Ad-wtBik- or Ad-{Delta}Bik-infected HT-29 cells (Fig. 2), indicating that mitochondrial toxicity was attributable to the BH3 domain of Bik and not related to the adenoviral construct itself. Furthermore, infection of cells with the empty cassette vector control adenoviral construct had no effect (results not shown).



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  		Fig. 1 Requirement of the BH3 domain for Bik-mediated mitochondrial toxicity. PC-3 and HT-29 cells were infected with graded concentrations of Ad-wtBik ({diamond} and {diamondsuit}) or Ad-{Delta}Bik ({square} and {blacksquare}). The effect of Ad infection on mitochondrial activity was determined by the colorimetric MTT assay 48 h postinfection. Each point represents a mean value of six measurements ±SD.

 


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  		Fig. 2 Exogenous expression of Bik in HT-29-infected cells. Logarithmically growing HT-29 cells were infected with 250 pfu/cell of Ad-wtBik or Ad-{Delta}Bik. Cells were harvested 48 h postinfection and lysed in sample loading buffer. Equal amounts of proteins (20 µg) were electrophoresed on a 12% SDS-PAGE and transferred onto Hybond-C membranes. In A, expression of adenoviral Bik was detected using an anti-HA antibody. In B, the same blot was reprobed with a cleaved caspase-7 antibody (D198) and a cleaved PARP antibody (D214), both from Cell Signaling Technology.

 
BH3-dependent Bik Toxicity Is Associated with Apoptosis
To determine whether the effect of Bik on mitochondrial activity was associated with apoptosis, we treated PC-3 and HT-29 cells with either Ad-wtBik or Ad-{Delta}Bik for 48 h and fixed cells for Hoechst staining. As shown in Fig. 3, A and C, nuclei from PC-3 and HT-29 cells treated with Ad-wtBik were condensed and pyknotic, whereas such morphological changes were not observed in Ad-{Delta}Bik-infected cells (Fig. 3, B and D). Because a characteristic feature of apoptosis is the presence of internucleosomal DNA fragmentation, PC-3 and HT-29 cells infected with the Ads were further analyzed for DNA fragmentation in agarose gels. The characteristic DNA ladder was present only in cells treated with the wt BikAd construct and not with BH3-deleted BikAd construct (Fig. 4).



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  		Fig. 3 Induction of apoptosis by Ad-mediated overexpression of Bik is dependent on its BH3 domain. PC-3 (A and B) and HT-29 (C and D) cells were infected with 250 pfu/cell of Ad-wtBik (A and C) or Ad-{Delta}Bik (B and D). Postinfection (24 h), the cells were fixed and stained using Hoechst 33343 as described in "Materials and Methods." Apoptotic bodies (arrows) were observed only in Ad-wtBik-treated cells (A and C). Magnification: x40.

 


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  		Fig. 4 DNA fragmentation. PC-3 and HT-29 cells were infected with 250 pfu/cell of Ad-wtBik or Ad-{Delta}Bik. After 48 h, cells were harvested, and genomic DNA was analyzed by agarose gel electrophoresis. DNA laddering was present in Ad-wtBik-treated cells only. M, 1-kb DNA ladder.

 
BH3-dependent Bik Induction of Apoptosis Is Accompanied by Cytochrome c Release and Caspase Activation
To further investigate the mechanism of apoptosis associated with Ad-wtBik infection, we evaluated cytochrome c release. PC-3 and HT-29 cells were infected with both Ad constructs for 24 and 48 h. As shown in Fig. 5, cytochrome c was present only in cytosolic extracts obtained from cells infected with wt BikAd construct. When cells were infected with the Bik gene deleted in the BH3 domain, cytochrome c was not detected in the cytosolic extracts. The release of cytochrome c from the mitochondria to the cytosol is believed to act as a trigger of apoptosis by activating different initiator caspases, including caspases 8, 9, and 10 (20-22), which in turn cleave and activate downstream executioner caspases 3, 6, and 7, leading to the degradation of essential cellular components (23-25). Therefore, we used antibodies that specifically recognize active, cleaved caspases 9, 7, and 3 to monitor the effect of overexpression of Bik on caspase cleavage. Activation of caspases was time dependent, with more caspases cleaved after a 48-h treatment compared with 24 h (Fig. 6) and also dependent on the BH3 domain. Whereas exogenous Bik levels were similar in Ad-wtBik- and Ad-{Delta}Bik-infected cells (Fig. 2), we could not detect any caspase cleavage products in cells infected with the Ad-{Delta}Bik (Figs. 2 and 6). Finally, we analyzed the state of the endogenous apoptotic substrate, PARP (26). During apoptosis, PARP, an Mr 116,000 nuclear poly (ADP ribose) polymerase is cleaved by caspase 3, giving rise to a fragment of Mr 24,000 (NH2-terminal DNA binding domain) and a fragment of Mr 89,000 (COOH-terminal catalytic domain; Refs. 27 and 28). Using a cleaved PARP (D214) antibody, the Mr 89,000 fragment of PARP was present only in Ad-wtBik-infected cells in which caspases were activated (Figs. 2 and 6).



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  		Fig. 5 Ad-wtBik-induced apoptosis is accompanied by the release of cytochrome c. PC-3 (1 x 107 cells) and HT-29 (0.5 x 107 cells) cells were treated with Ad-wtBik or Ad-{Delta}Bik. Cells were harvested after 24 or 48 h of infection, and cytosolic extracts were prepared as described in "Materials and Methods." Equal amounts of proteins (10 µg) were electrophoresed on SDS-PAGE and transferred to Hybond-C membranes. The membrane was probed with a mouse monoclonal antibody specific for human cytochrome c. The blot was stripped and then reprobed with an antibody against ß-actin to verify that the protein loading was equal in all lanes. CTR, uninfected control cells.

 


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  		Fig. 6 Ad-wtBik cytochrome c release is accompanied by caspase activation and PARP cleavage. PC-3 (left panel; 1 x 107 cells) and HT-29 (right panel; 0.5 x 107 cells) cells were treated with Ad-wtBik or Ad-{Delta}Bik. Cells were harvested after 24 or 48 h of infection and lysed in sample loading buffer. Equal amounts of proteins (20 µg) were electrophoresed on SDS-PAGE and transferred to Hybond-C membranes. Cleaved caspases 9, 7, and 3 and PARP products were revealed by immunoblotting with enhanced chemiluminescent detection. ß-actin was used to verify loading of similar amounts of cell lysates.

 
Ad-wtBik Causes Tumor Regression in PC-3 and HT-29 Xenograft Tumor Models
The potent in vitro apoptotic activity of Bik led us to evaluate its antitumor properties. CD1 male nude mice (6–8 weeks old) were injected s.c. with PC-3 cells. Mice received two intratumoral injections of 2 x 109 or 20 x 109 pfu of Ad-{Delta}Bik or Ad-wtBik at days 21 and 23 after tumor cell inoculation, once tumors had reached a palpable size (~40 mm3). Treatment with either dose of Ad-wtBik resulted in significant tumor regression occurring around day 26 (Fig. 7, left panel). When animals were sacrificed at day 35, because of necrotic tumors in the control groups, the percentage of tumor growth inhibition was 86 and 93% for the 2 x 109 and 20 x 109 pfu q2d x 2 Ad-wtBik-treated groups, respectively. On the other hand, animals whose tumors were injected with Ad-{Delta}Bik showed tumor growth curves similar to the saline-treated group (Fig. 7, left panel). These results indicate that overexpression of Bik leads to tumor regression and that the antitumor activity of Bik is dependent on its BH3 domain. Furthermore, intratumoral injection of the Ads did not induce any systemic toxicity or weight loss (data not shown).



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  		Fig. 7 Requirement of BH3 for antitumor activity of Bik. Left panel, male CD1 nu/nu mice were injected s.c. with 2 x 106 PC-3 cells on day 0. Treatment with Ad-wtBik or Ad-{Delta}Bik was begun once tumors were established. Mice received two intratumoral injections, on days 21 and 23. Right panel, female CD1 nu/nu mice were injected s.c. with 2 x 106 HT-29 cells on day 0. Treatment with Ad-wtBik or Ad-{Delta}Bik was started once tumors were established. Mice received a total of four intratumoral injections, on days 9, 11, 14, and 16. Tumor volumes were measured in six to seven mice every other day for >30–35 days, and data represent the means ±SE.

 
HT-29 cells were more sensitive to overexpression of Bik than PC-3 cells in vitro, and tumors of the HT-29 xenograft model exhibit faster growth than PC-3 xenografts. Therefore, we evaluated the antitumor effect of Bik on this more aggressive and refractory tumor xenograft model using multiple injections of both Ad constructs. In this model, palpable tumors (average volume 80 mm3) were obtained 7 days after tumor cell inoculation. Mice received two intratumoral injections at days 9 and 11, and when no obvious regression was observed, two additional injections were given at days 14 and 16. Thus, each mouse received a total of four injections of 15 x 109 pfu of Ad-{Delta}Bik or Ad-wtBik. Tumors regressed after the fourth injection (Fig. 7, right panel), and on sacrifice, the mean tumor growth inhibition was 92%, with four of seven animals having tumors <12 mm3 at the time of sacrifice (Fig. 8, left panel). Similar to the results obtained in PC-3 xenografts, tumors injected with Ad-{Delta}Bik grew similarly to saline-treated controls, resulting in 860% increase in tumor volume (Fig. 7).



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  		Fig. 8 Induction of apoptosis by overexpression of Bik in HT-29 tumors requires the BH3 domain. HT-29 s.c. tumors in the right flank of nude mice after treatment with Ad-{Delta}Bik (A) or Ad-wtBik (B). Arrows, the location of tumors treated with Ad-wtBik. C, histology of tumor sections: H&E (1), TUNEL (2), and Hoechst (3) staining of HT-29 tumors after two daily injections of Ad-wtBik, x40 magnification. 4, H&E staining of Ad-{Delta}Bik-treated tumors, x40 magnification.

 
In Vivo Antitumor Activity of Bik Is Associated with Apoptosis
Microscopic observation of HT-29 tumor sections after H&E staining showed that massive cell death occurred in the tumors treated with the Ad-wtBik (Fig. 8, right panel, section 1). The cell death was characterized by a high number of irregular and small nuclei (Fig. 8, right panel, sections 1 and 3) that were positive for TUNEL staining (Fig. 8, right panel, section 2), indicating that tumor regression after Ad-wtBik injection was associated with apoptosis. These morphological changes or TUNEL-positive cells were not observed in Ad-{Delta}Bik-injected tumors (Fig. 8, right panel, section 4) or saline controls (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent studies have demonstrated that Ad-mediated overexpression of the pro-apoptotic Bak (15) and Bax genes (13, 14, 29) resulted in tumor regression. Bak and Bax are apoptotic-inducing proteins that contain BH regions 1, 2, and 3 (30, 31). It is the BH3 domain of these pro-apoptotic Bcl-2 members that provide their apoptotic activity. Indeed, deletion mutant analysis of Bak has demonstrated that loss of BH3 prevents its death-inducing properties (7). Furthermore, a 15mer synthetic peptide of the Bak protein, when delivered to cells, was sufficient to cause apoptosis (10). The identification of apoptotic Bcl-2 family members containing only the BH3 domain, such as Bad (32, 33), Bid (34), Bik (12), and Noxa (35), further documents the importance of the BH3 domain in apoptosis.

The present study was performed to identify the pro-apoptotic signal pathway of the Bik protein in both PC-3 and HT-29 cells in vitro and provide new evidence of its potent antitumor activity in human tumor xenografts. To study the pro-apoptotic activity of the Bik protein, the Bik cDNA was constructed in an Ad delivery system that displays a high rate for both infection and expression of proteins in mammalian cells, thus providing an advantage for both in vitro and in vivo studies. Ectopic expression of Bik in PC-3 and HT-29 cells induced a loss of mitochondrial activity in 60 and 90% of the cells, respectively, 48 h after virus infection. The loss of mitochondrial activity measured by the MTT assay was associated with apoptosis as shown by characteristic cellular morphological changes and DNA fragmentation. A Bik mutant, lacking the BH3 domain, was totally inactive, consistent with previous studies indicating that Bik promotes apoptosis via its BH3 domain (12, 16). However, the detailed mechanism by which Bik induces apoptosis is not well understood. It has been reported that Bik causes apoptosis in MCF-7 human breast cells downstream of the CrmA block but upstream of distal caspases (36). When we overexpressed Bik in PC-3 and HT-29 tumor cell lines, we observed the release of cytochrome c from the mitochondria to the cytoplasm. The cytochrome c release led to the activation of caspases 9, 7, and 3, which were shown to be induced in a time-dependent fashion. Cleavage products of these caspases were not observed in cells infected with the BH3-deleted Bik gene. Finally, we analyzed the state of the endogenous caspase 3 substrate, PARP (26) Overexpression of wt Bik protein led to the appearance of the apoptosis-specific Mr 89,000 PARP fragment. The Mr 89,000 PARP fragment was not detected in cells overexpressing the BH3-deleted protein despite the fact that comparable Bik protein expression levels were achieved using the wt and deleted BH3 adenoviral constructs.

Clinical trials for Ad-mediated gene therapy for cancer treatment are currently ongoing with the tumor suppressor p53 gene and the herpes thymidine kinase gene (37-40). Thus, Ad-mediated gene transfer is clinically relevant and has been shown to induce apoptosis of tumor cells while sparing normal adjacent cells (41). With that precedent in mind, we investigated whether the apoptosis-inducing function of Bik could effectively mediate tumor growth suppression in vivo. Intratumoral injection of adenoviral wt Bik led to tumor regression in both models tested. Because PC-3 cells are particularly resistant to apoptosis (42, 43) and HT-29 xenografts are refractory to current chemotherapeutic agents (44), our results suggest that the Bik gene may be an alternative to current antineoplastic treatment. Massive numbers of apoptotic bodies, characterized by Hoechst staining and TUNEL-positive cells, were observed in the tumor sections obtained from wt Bik-treated tumors. As apoptotic bodies are rarely observed in vivo because of a rapid clearing by macrophages (45), the accumulation of apoptotic cells in wt Bik-treated tumor sections is a further indication of the potent and rapid cell death-inducing activity of Bik.

In summary, the proapoptotic mechanism of Bik on PC-3 and HT-29 cells was studied both in vitro and in vivo, and the results suggest that Bik promotes cell apoptosis through a mitochondrial-associated caspase pathway. Our results demonstrated that the Bik gene can effectively induce apoptosis and suppress tumor growth both in vitro and in vivo. Furthermore, we showed that the antitumor effect of Bik is dependent on its BH3 domain.


   Acknowledgments
 
We thank Lucie Bibeau and Chantal Boudreau for their excellent technical assistance in animal care, David Bouffard for helpful discussions, and Johanne Cadieux for her assistance with the preparation of the manuscript.


   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 indi-cate this fact.

1 Supported by research Grants CA-73803 and CA-33616 from the National Cancer Institute (to G. C.). Back

2 To whom requests for reprints should be addressed, at Shire BioChem, Inc., 275 Boulevard Armand-Frappier, Laval, Québec, H7V 4A7 Canada. Email: hgourdeau{at}ca.shire.com Back

3 The abbreviations used are: BH, Bcl-2 homology domain; wt, wild type; Ad, adenovirus; pfu, plaque forming unit(s); TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; PARP, poly(ADP-ribose) polymerase; MTT, 3-(4,5-dimethylthiazyl-2-yl)-2,5-diphenyltetrazolium bromide; HA, hemagglutinin. Back

Received 7/18/01; revised 9/17/01; accepted 9/18/01.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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