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Annals of Surgical Oncology 10:314-322 (2003)
© 2003 Society of Surgical Oncology

ORIGINAL ARTICLES

The Mitochondrial Apoptosis-Inducing Factor Plays a Role in E2F-1–Induced Apoptosis in Human Colon Cancer Cells

Stephan A. Vorburger, MD, Abujiang Pataer, MD, PhD, Kazumi Yoshida, MD, Yanna Liu, MS, BS, Xiaolin Lu, MS, BS, Stephen G. Swisher, MD and Kelly K. Hunt, MD

From the Departments of Surgical Oncology (SAV, YL, XL, KKH) and Thoracic and Cardiovascular Surgery (AP, SGS), University of Texas, M. D. Anderson Cancer Center, Houston, Texas; and the Department of Surgery (KY), Jikei University School of Medicine, Tokyo, Japan.

Correspondence: Address correspondence and reprint requests to: Kelly K. Hunt, MD, FACS, Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 444, Houston, TX 77030; Fax: 713-792-4689; E-mail: khunt{at}mdanderson.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Overexpression of the transcription factor E2F-1 provokes apoptosis in cancer cells; the mechanism, however, is not completely understood. We sought to evaluate E2F-1 gene therapy in human colon cancer and to investigate the apoptotic pathway involved.

Methods: Adenoviral vectors were used to transfect the E2F-1 gene (Ad5E2F-1) or the control gene luciferase (Ad5Luc) into four human colon carcinoma cell lines. Apoptosis was confirmed by flow cytometry and poly (ADP-ribose) polymerase cleavage. Expression of apoptotic factors was determined with Western blot analysis. Inhibitory assays were used to determine the involvement of caspases in the apoptotic pathway.

Results: Overexpression of E2F-1 was evident in all cells treated with Ad5E2F-1; upregulation of Bcl-2, and activation of caspases were noted. The apoptosis-inducing factor in the cytosolic fraction was markedly upregulated after Ad5E2F-1 treatment. E2F-1 overexpression inhibited proliferation and induced significant apoptosis in all cell lines (P < .005). This apoptotic response could be only partially blocked by caspase inhibitors.

Conclusions: These findings demonstrate that E2F-1 induces apoptosis and inhibits proliferation in human colon cancer cell lines. The marked upregulation of apoptosis-inducing factor and the fact that E2F-1–induced apoptosis is incompletely blocked by caspase inhibitors suggest a caspase-independent pathway of E2F-1–mediated apoptosis, reported here for the first time.

Key Words: Apoptosis • E2F-1 • Colon cancer • Apoptosis-inducing factor • Mitochondrion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis (programmed cell death) is a mechanism of cell death through which multicellular organisms eliminate unwanted cells to ensure proper development and maintain cellular homeostasis.1,2 One of the hallmarks of cancer cells is their resistance to proapoptotic stimuli. Most of the anticancer drugs currently in use mediate their effect through the induction of apoptosis, and resistance to these chemotherapeutic agents is often due to mutations in the programmed cell-death pathway.3–5 The sequence through which polyps develop into colorectal tumors involves mutations in >70% of genes relevant for the apoptotic response.6,7 Therefore, therapeutic strategies that allow the cells to overcome this apoptotic block could prove valuable in chemoresistant colorectal cancers.

E2F-1 is a member of a family of transcription factors that play a key role in control of cell proliferation by linking the activities of the cell-cycle machinery with the transcriptional regulation of genes required for S-phase entry and DNA synthesis. Activation of the E2Fs by release from the complex with the retinoblastoma protein induces expression changes of >1200 genes.8 Although most of the E2F members can induce proliferation and differentiation, only E2F-1 is known to effectively induce a broad variety of tumor cells to undergo programmed cell death.9–17 To date, the mechanism by which E2F-1 induces apoptosis is not completely understood, but ultimately caspases consisting of initiators and effectors are sequentially activated to form a proteolytic cascade to induce DNA fragmentation and cell death.18–20 Two main caspase-activating cascades have been characterized: the mitochondrial-mediated caspase-3 activation by caspase 9 (intrinsic pathway) and the death-receptor–induced caspase-3 activation by caspase 8 (extrinsic pathway).21

Apoptosis through the mitochondrial pathway is triggered by the release of cytochrome c from the intermembrane space of the mitochondrion. Cytochrome c then complexes with apoptotic protease activating factor-1 (Apaf-1) (apoptosome) to cleave pro–caspase 9 into active caspase 9. E2F-1 overexpression has been reported to induce upregulation of Apaf-1 and pro–caspase 3, 6, and 7, but the nonapoptotic family members E2F-2 and E2F-3 induced similar protein levels.8

Recently, the mitochondrial apoptosis-inducing factor (AIF) has been described to be an important molecule in caspase-independent killing.22 AIF can induce chromatin condensation and large-scale DNA fragmentation in a caspase-independent manner.23,24 Like cytochrome c, AIF is located in the intermembrane space of the mitochondrion, where it is cleaved and folded into its active form. Opening of the mitochondrial permeability transition pores (PTP), which are under the control of members of the Bcl-2 family, causes the release of these soluble proteins from the intermembrane space.25 Bcl-2 inhibits the mitochondrial release but has no cytoprotective effect once AIF is present in the cytosol. Therefore, it is likely that AIF acts beyond or independently of the Bcl-2 and caspase checkpoints in the cell-death process.

We evaluated the role of caspase-independent apoptosis through AIF in the response to adenovirus-mediated overexpression of E2F-1 in colon cancer cells. Although mutations of the caspases are rarely found in tumors, recent studies have convincingly demonstrated that deregulated expression of apoptosis-mediating factors may confer drug resistance.26,27

We found that E2F-1 induces cell death in human colon cancer cells through both caspase-independent and caspase-dependent pathways. This could make E2F-1 a promising transgene in gene therapy approaches against multidrug-resistant colorectal cancers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Reagents
The primary human colon carcinoma cell line SW480 and the metastatic human colon cancer cell lines SW620 and HT-29 were obtained from the American Type Culture Collection (Manassas, VA). The metastatic colon carcinoma cell line KM12L4 was kindly provided by Dr. Lee M. Ellis (M. D. Anderson Cancer Center, Houston, TX). Cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum supplemented with 2 mM of L-glutamine, 100 U/mL of penicillin, and 100 µg/mg of streptomycin. Cells were treated at a confluence of 75% to 85%.

Adenovirus Vectors
We used replication-deficient human type 5 adenoviruses with the E1A region replaced with E2F-1 (Ad5E2F-1), the luciferase reporter gene (Ad5Luc), or the ß-galactosidase reporter gene (Ad5ßGal) under the control of the cytomegalovirus promoter. Adenoviral constructs were tested for E1A contamination by using reverse transcriptase-polymerase chain reaction. Electron microscopy of infected cells was used to confirm the lack of viral replication. Viral particles (vp) were measured with optical densitometry; plaque-forming assays using 293 cells were performed as described.28 A multiplicity of infection of 200 plaque-forming units per cell led to transduction of approximately 85% to 90% of the cells as determined by ß-galactosidase expression after infection with Ad5ßGal.

Flow Cytometry Analysis
We measured apoptotic cells by using propidium iodide staining and fluorescence-activated cell sorter (FACS) analysis. Supernatant and plated cells were harvested, pelleted by centrifugation, and resuspended in binding buffer (phosphate-buffered saline [PBS], .1% Triton X-100 [Rohm & Haas Co., Philadelphia, PA], and .1% sodium citrate) with a final propidium iodide concentration of 50 µg/mL. Cells were incubated at 4°C for 16 hours in the dark. Each sample was analyzed in triplicate by using FACS (FACScan; Becton-Dickson, Mountain View, CA; FL-3 channel). Cells with a DNA content less than G1 (subdiploid) were considered to undergo apoptosis.

DNA Fragmentation Assay
DNA was isolated with a phenol-chloroform extraction procedure. The genomic DNA was analyzed for degradation by using gel electrophoresis. The agarose gels were photographed and examined for evidence of DNA oligonucleosomal fragmentation (DNA laddering), which confirms apoptosis.

Western Blot Analysis
Whole-cell lysates were collected from adherent and floating cells after incubation of tissue culture with triple lysis buffer for 20 minutes at 4°C and subsequent centrifugation at 14,000 rpm. Protein concentration was measured with the Bio-Rad assay (Bio-Rad, Hercules, CA), and 20 µg of protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Antibodies used and their sources were as follows: E2F-1 (KH95); AIF (H-300); Bcl-2; Bak (N-20); Bax (P-19); tumor necrosis factor (TNF)-receptor 1 (R1) (H-271); TNF-{alpha} (H-156); TNF-R1 associated death domain (TRADD) (H-278) (Santa Cruz Biotechnology; Santa Cruz, CA); cytochrome c; caspase 3, caspase 8, and caspase 9 (PharMingen, San Diego, CA); and ß-actin (Sigma-Aldrich Chemicals, St. Louis, MO). Membranes were then incubated with secondary horseradish peroxidase-conjugated antibodies (anti-goat immunoglobulin G, Santa Cruz Biotechnology; anti-rabbit and anti-mouse immunoglobulin G, Sigma-Aldrich Chemicals) for 1 hour at 4°C. The membranes were developed according to Amersham’s electrogenerated chemiluminescence protocol (Amersham, Arlington Heights, IL).

Western Blot Analysis of Fractionated Cytosolic Cell Extract
The release of AIF from mitochondria was measured by immunoblotting as described previously. Cells were harvested by centrifugation and gently lysed for 5 minutes in an ice-cold buffer containing 25 mM of Tris and 5 mM of MgCl2, pH 7.4. Lysates were centrifuged for 5 minutes at 16,000 x g, the protein concentration of the supernatant was determined by the Bio-Rad method (see above), and immunoblotting was performed as described previously.

Caspase Inhibitory Assays
Cell cultures were pretreated with caspase inhibitors 1 hour before infection with Ad5E2F-1 or controls and again 18 hours after infection. Irreversible, cell-permeable caspase inhibitors were dissolved in dimethyl sulfoxide in a 50 mM stock solution. Final dimethyl sulfoxide concentrations in culture media were well below toxic levels of .2%. Preliminary experiments were conducted to titrate optimal concentrations of caspase inhibitor. Toxicity was excluded by preliminary studies with negative control caspase inhibitor (Z-VAD-FMK). The following concentrations were used: caspase-9 inhibitor, Z-LEHD-FMK at 50 µM; caspase-8 inhibitor, Z-IETD-FMK at 50 µM; and pan-caspase inhibitor, Z-VAD-FMK at 50 µM (all caspase inhibitors from BD PharMingen).

Statistical Analysis
If not mentioned otherwise, error bars represent 1 SE of triples of independent experiments. P values were calculated with Student’s two-tailed t-test for two-sample unequal variance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
E2F-1 Induces Cell-Cycle Progression in Cancer Cells
To determine the transduction efficiency of our adenoviral vector, we used an identical vector construct that carried the ß-galactosidase reporter gene under the control of the cytomegalovirus promoter. Because adhesion and integration of adenovirus type 5 depend on the expression of the Coxsackie-adenovirus receptor and on the presence of {alpha}vß3-integrin of the host cells, transduction efficiency varies between cell lines and is not determined by the transgene inserted. Efficient transduction could be detected at a multiplicity of infection of <1600 vp per cell in all cell lines tested (Fig. 1).



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  		FIG. 1. Measurement of transduction efficiency 48 hours after treatment of the different cell lines with increasing multiplicity of infection (MOI) in viral particles (vp) per cell with an adenoviral vector carrying the ß-galactosidase gene (AdßGal). Transfected cells stain blue after exposure to X-galactosidase, which allows the quantification of transfected cells by light microscopy. The transduction rate of adenoviral vectors depends on the cell type rather than on the transgene inserted, thus allowing extrapolation of the efficiency of transduction of the same vector that carries a therapeutic gene.

 
At 36 hours after infection with Ad5E2F-1 at a multiplicity of infection of 2000 vp per cell, cells were harvested and stained for DNA content with propidium iodide. In E2F-1–treated cultures, flow cytometric analysis of the cell cycle showed a decrease in the fraction of cells in G1 phase and an increase of the cell fraction at the G2/M checkpoint (Fig. 2A). This correlates with the known function of E2F-1 to promote cell-cycle progression from G1 to S phase. Colon cancer cells treated with PBS or Ad5Luc did not show any significant changes in the cell-cycle profile.



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  		FIG. 2. (A) Flow cytometric analysis of cells stained with propidium iodide. Measuring the amount of DNA in each cell allowed quantification of cells in each phase of the cell cycle. Resting cells have a significant amount of DNA in the G1 phase, whereas cells in G2 phase have doubled their DNA content. Cells that have a DNA content less than G1 phase (DNA fragmentation) are undergoing apoptotic cell death. The dotted line to the left of G1 phase corresponds to the number of subdiploid (apoptotic) cells. Ad5E2F-1–treated cells are pushed into the cell cycle and show an increase in the proportion of cells in G2 phase (G2 arrest) and in apoptosis compared with phosphate-buffered saline (PBS)–treated or Ad5Luc-treated cell cultures. The table gives the percentage of the cells in each phase (±1 SD). (B) Quantification of apoptotic, but not necrotic, cell death with flow cytometry analysis of different colon cancer cell lines 48 hours after infection with Ad5E2F-1, Ad5Luc, or PBS. A significant increase in apoptosis of more than four times control levels was seen in all cell lines treated (results are the average of triplets representative of at least three independent experiments, and error bars show SE). The multiplicity of infection was 2000 viral particles per cell. Western blot analysis with E2F-1 monoclonal antibody confirmed corresponding overexpression of E2F-1 in all Ad5E2F-1–treated cells.

 
Ad5E2F-1 Infection Induces Overexpression of E2F-1 and Apoptosis in Human Colon Cancer Cell Lines
Western blot analysis of whole-cell lysates showed markedly increased levels of E2F-1 protein expression in all cell lines treated with Ad5E2F-1, but not in controls (Fig. 2B). E2F-1 overexpression was evident within 12 hours after infection with Ad5E2F-1. Flow cytometry was used to analyze the number of cells with a DNA content of less than G1 phase (subdiploid population). Subdiploid DNA content is seen when DNA is fragmented and degraded, a hallmark of apoptosis. Hence, FACS analysis can differentiate between those two forms of cell death. Forty-eight hours after infection with the Ad5E2F-1 vector, all colon cancer cell lines showed a significant increase in the subdiploid cell population compared with control treated cells (Fig. 2B). It is of interest to note that the primary colon carcinoma cell line SW480 and its metastatic counterpart SW620 were equally sensitive to E2F-1–induced apoptosis. Apoptotic cell death was further confirmed by Southern blot analysis for DNA oligonucleosomal fragmentation (DNA laddering) and by dark field microscopy, which showed morphological changes in the cells consistent with apoptotic cell death (data not shown).

E2F-1 Overexpression Inhibits Cell Proliferation
Growth curves of SW620, SW480, KM12L4, and HT-29 human colon carcinoma cells showed significantly inhibited proliferation and increased cell death after treatment with Ad5E2F-1 (Fig. 3). All cell lines showed some toxicity and decreased proliferation after treatment with Ad5Luc. Because not all of the cells will be effectively transduced and therefore overexpress E2F-1, the inhibition in cell growth seen in our experiments is remarkable and may be due to additional cytotoxic effects of the infected cells on noninfected neighboring cells.



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  		FIG. 3. Growth curves of colon cancer cell cultures. Twenty-four hours after the cells were plated at the same concentration, cultures were treated with Ad5E2F-1, Ad5Luc, or phosphate-buffered saline (PBS) at a multiplicity of infection of 2000 viral particles per cell. The number of adherent cells (living cell portion) was quantified by an automated cell counter gated to the size of the individual cell lines. The difference in the absolute initial cell count depended on the size and growing characteristics of cell lines. Ad5E2F-1 showed significant inhibition of proliferation in all four colon cancer cell lines within 48 hours.

 
E2F-1 Overexpression Induces the Caspase Cascade and Poly (ADP-ribose) Polymerase (PARP) Cleavage
Western blot analysis of whole-cell lysates of SW620 cells 48 hours after treatment with Ad5E2F-1, Ad5Luc, or PBS was performed (Fig. 4). Protein levels of the extrinsic caspase cascade (death-receptor pathway), which involves the receptor for TNF-{alpha} (TNF-R1), TRADD, and FAS-associated death domain, were not altered compared with controls. However, in E2F-1–treated cells, we noticed a slight downregulation of pro–caspase 8, which might indicate cleavage to its active form. Apparent cleavage of caspase 9 and caspase 3 correlates with activation of the mitochondrial pathway of apoptosis. PARP cleavage was demonstrated, confirming that cells were undergoing apoptosis in the Ad5E2F-1–infected cultures. We did not find any change in expression levels of pro–caspases 6 and 7. Thus, we cannot exclude that these caspases, which can compensate for caspase-3 loss, are not activated in E2F-1–overexpressing cells.



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  		FIG. 4. Western blot analysis of whole-cell lysates for various key proteins related to apoptosis and the caspase cascade (for the relevance of each protein, see Fig. 6). In addition to E2F-1 overexpression, other changes compared with controls were downregulation of pro–caspases 8, 9, and 3 and upregulation of Bcl-2. Cleavage of caspase 3 and poly (ADP-ribose) polymerase (PARP) is a hallmark of apoptosis. ß-Actin served as a control for equal protein loading. In the column to the right of the Western blots, the integrated density values, as measured by densitometry of the corresponding protein expression levels, are listed. Western blots are representative of several independent experiments. PBS, phosphate-buffered saline; TNF, tumor necrosis factor; TRADD, TNF-R1 associated death domain.

 


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  		FIG. 6. Schematic drawing of the apoptotic pathway involved in E2F-1–induced apoptosis. The release of apoptosis-inducing factor (AIF) from the intermembrane space of the mitochondrion allows for a caspase-independent induction of apoptosis. This mechanism seems to be involved in E2F-1–induced apoptosis in colon cancer cells.

 
Bcl-2 Family Members
Bid is a member of the Bcl-2 family, which is cleaved by caspase 8 into its proapoptotic form tBid. tBid and Bax induce mitochondrial membrane destabilization, permitting cross-talk between the two caspase cascades. Whereas Western blot showed no variations in Bid or Bax levels, Bcl-2 expression was clearly upregulated (Fig. 4). These findings, which are consistent in all of the cell lines tested, are surprising because Bcl-2, an antiapoptotic mediator, is known to inhibit the opening of the mitochondrial PTP and to stabilize the mitochondrial membrane.

E2F-1–Induced Apoptosis Can Be Only Partially Blocked by Caspase Inhibitors
Caspase activation can be selectively and irreversibly inhibited by pretreatment of the cell cultures with Z-LEHD-FMK (blocks caspases 4, 5, and 9), Z-IETD-FMK (blocks caspase 8 and Granzyme B), or the total caspase inhibitor Z-VAD-FMK. Even after titration for optimal dosage of caspase inhibitors, we were not able to completely block E2F-1–induced apoptosis. The decrease in the apoptotic response specific to Ad5E2F-1 treatment was 70% after caspase inhibition (Fig. 5).



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  		FIG. 5. Quantification of apoptotic cells by flow cytometry analysis of propidium iodide–stained SW620 cells 48 hours after infection with phosphate-buffered saline (PBS) or Ad5E2F-1. Cells either were not pretreated or were preincubated 1 hour before infection with 40 µmol of caspase-9 inhibitor, with caspase-8 inhibitor, or with a pan-caspase inhibitor. After 18 hours, caspase inhibitors were readministered. The apoptotic response to Ad5E2F-1 could be reduced only approximately 60% with pan-caspase inhibitor (pretreatment of a higher dose of caspase inhibitors resulted in increased toxicity, as determined by preliminary results). Corresponding Western blot analysis and quantification with densitometry of the cytoplasmic protein fraction with anti–apoptosis-inducing factor (AIF) antibody shows AIF release from the mitochondrion. Decreased cytoplasmic AIF protein after pan-caspase–inhibitor or caspase-8–inhibitor treatment could point to the involvement of Bid-mediated caspase-8 activation of mitochondrial release.

 
AIF Is Released After Ad5E2F-1 Infection
By separating the cytosolic protein fraction from the mitochondria, Western blot analysis allowed us to verify that AIF is released into the cytoplasm in response to E2F-1 overexpression. Not surprisingly, we found significant amounts of AIF in the cytosol in Ad5E2F-1–treated cells, but not in PBS- or Ad5Luc-treated controls (Fig. 5). The mitochondrial pathway involves the release of cytochrome c from the mitochondrium, which then binds to Apaf-1 to activate caspase 9. Because cytochrome c and AIF are both located in the intermembrane space, the opening of the PTP would also allow AIF to escape into the cytoplasm and then further migrate into the nucleus, where it can directly induce DNA fragmentation. The fact that AIF release is partially downregulated after pretreatment of the cells with caspase 8 or pan-caspase inhibitors is suggestive of an involvement of Bid/tBid in the E2F-1 apoptotic response (Fig. 6). However, the residual amounts of cytoplasmic AIF found in caspase-inactive cells are sufficient to induce apoptosis independent of the caspase cascade.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
E2F-1 Induced Apoptotic Cell Death in Human Colon Cancer Cells
In this study, we induced E2F-1 overexpression in colon cancer cell lines by using an adenoviral vector system. We demonstrated that E2F-1–mediated apoptosis occurs despite upregulation of the antiapoptotic mediator Bcl-2 and that release of the AIF from the mitochondrium is involved. These findings point to a caspase-independent pathway through which the apoptotic response to E2F-1 overexpression is mediated.

Our data support previous studies that showed that adenoviral-mediated overexpression of the transcription factor E2F-1 induces apoptosis in a variety of carcinoma cell lines, as well as colon cancer cell lines.9,10,12–18,20,29–33 The findings reported here extend these previous studies of the effect of E2F-1 on primary and metastatic colon cancer cell lines and contribute to the understanding of E2F-1–induced apoptosis.

E2F-1–Induced Apoptosis Is Mediated by the Activation of the Caspase Cascades
One of the key components of tumor suppression in many cell types is the p53 protein, which contributes to the apoptotic elimination of cells undergoing oncogenic stress.34–36 Overexpression of E2F-1 induces the accumulation of p53, and it was originally believed that p53 was essential for E2F-1–mediated apoptosis.37,38 This assumption was challenged by a number of studies that reported E2F-1–induced cell death in cell lines with p53 mutations or p53 deletions.13,18,39,40 However, recent findings by Irwin et al.19 introduced the p53 homolog p73, which allows a potential compensatory pathway for E2F-1–induced apoptosis in cells with mutated p53. In this study, one primary and three metastatic human colon cancer cell lines were examined, all of which have mutations in p53. Adenovirus-mediated overexpression of E2F-1 effectively induced apoptotic cell death in all cell lines tested.

Ad5E2F-1 Induced Mitochondrial Release and Apoptosis Despite Upregulation of Bcl-2
Bcl-2 is one of the best-studied antiapoptotic proteins. It has been shown to block the mitochondrial pathway of apoptosis, which depends on cytochrome-c release, on the initiator caspase-9 and the execution caspases-3 and -7.41,42 The connection between E2F-1 and Bcl-2 was established after the discovery that extrinsic Bcl-2 overexpression can protect cells from E2F-1–mediated apoptosis.43 We found that adenoviral-mediated overexpression of E2F-1 increased Bcl-2 levels significantly in colon cancer cell lines. These findings support recent publications that show that E2F-1 upregulates transcription of Bcl-2 in most cell lines.8,44,45 In contrast to reports by Yang et al.,46 we did not find that levels of Bcl-2 expression correlated with the extent of apoptosis. We did not, however, determine the levels of phosphorylated (inactivated) Bcl-2, which was shown to have the capability to inactivate its unphosphorylated form. Unlike E2F-2 and E2F-3 (which also upregulate Bcl-2 but cannot induce apoptosis), the induction of Bcl-2 by extrinsic overexpression of E2F-1 is apparently insufficient for counteracting the resultant apoptotic response. One could speculate, therefore, that the apoptotic response to E2F-1 is mediated mainly through the nonmitochondrial (death-receptor) pathway, which would not be affected by Bcl-2 levels. Interestingly, we found that despite the inhibition of the nonmitochondrial pathway by caspase 8 and pan-caspase inhibitors, AIF was released and apoptosis was observed. These results suggest that E2F-1 overexpression depends, at least in part, on the mitochondrial pathway to induce cell death and that E2F-1 can override the antiapoptotic activity of Bcl-2. E2F-1 gene therapy could therefore prove to be effective in Bcl-2–overexpressing tumors that are commonly resistant to chemotherapeutic agents.41,42,47,48

AIF Release Is Involved in E2F-1–Induced Cell Death
Apoptosis induced by overexpression of p53 or E2F-1 results in upregulation of c-myc, and it has been suggested that c-myc may mediate E2F-1–induced apoptosis in colon cancer.49,50 Apaf-1 and activation of caspase 9 seem to be required for both p53-dependent and c-myc–induced apoptosis, and deregulation of this apoptosome complex is associated with chemoresistance.51,52 The fact that in our experiments the blocking of the apoptosome activation, as well as the inhibition of all caspase activity, only partially abrogates E2F-1–induced apoptosis suggests that E2F-1 uses additional pathways that are independent of c-myc.

Other investigators have used staurosporine to induce the permeabilization of the outer mitochondrial membrane and subsequent release of AIF and reported that a pan-caspase inhibitor does not prevent AIF release from the mitochondrion.53 AIF translocation correlates with the appearance of large-scale DNA fragmentation; hence, it offers a caspase-independent apoptotic pathway.22,25 Our data confirmed that AIF is released after E2F-1 overexpression and that blocking of caspase activation only partially reduced the apoptotic response to AdE2F-1 treatment in colon cancer cells.

Here we demonstrate that gene therapy with adenovirus-mediated E2F-1 overexpression inhibits proliferation of human colon cancer cell lines by induction of apoptosis. Furthermore, we report for the first time that this apoptotic response involves the AIF and that it is only partially dependent on the activation of the caspase cascade. Therefore, therapeutic strategies that involve adenovirus-mediated E2F-1 gene therapy could prove valuable in the treatment of colon cancer metastases that are chemoresistant because of inactivation of the caspase cascades.


   Acknowledgments
 
The acknowledgments are available online at www.annalssurgicaloncology.org.


   Footnotes
 
E2F-1 induces apoptosis in colon cancer cells, and this can occur independently of caspase activation and involves mitochondrial release of the apoptosis-inducing factor. Understanding the pathways through which E2F-1 induces apoptosis may prove valuable for therapeutic approaches.

Received for publication May 16, 2002. Accepted for publication October 28, 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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