E2F-1 Up-Regulates C-Myc and P14arf and Induces Apoptosis in Colon Cancer Cells1

3590 Vol. 7, 3590–3597, November 2001 Clinical Cancer Research

E2F-1 Up-Regulates c-Myc and p14ARF and Induces Apoptosis in Colon Cancer Cells1

Mary Jane Elliott, Yan Bin Dong, Hailiang Yang, tion of Mcl-1. Therefore, E2F-1 is a potentially active gene and Kelly M. McMasters2 therapy agent for the treatment of colon cancer. Department of Surgery, University of Louisville, James Graham Brown Cancer Center, Louisville, Kentucky, 40202 INTRODUCTION Essentially all of the anticancer drugs that are currently ABSTRACT available as well as UV irradiation have been shown to kill Although overexpression of E2F-1 can induce apoptosis cancer cells by triggering apoptosis (1). Defective apoptotic in a variety of tumor cell lines, the mechanisms by which mechanisms are considered to play a role in both the develop- E2F-1 induces apoptosis remain ambiguous. In this study, ment of malignancy and resistance to a wide range of chemotherapeutic drugs (1, 2). Studies have shown that mutations in we examine the ability of E2F-1 to induce apoptosis in colon the genes involved in apoptosis are responsible for Ͼ70% of the cancer and the molecular mechanisms underlying E2F-1- polyp-tumor progression of colorectal cancer (3–5) and may mediated apoptosis. HT-29 and SW-620 colon adenocarci- contribute to the multidrug resistant phenotype of these cancers noma cells (both mutant p53) were treated by mock infec- (6). Therefore, the development of new treatments that can tion or adenoviral vectors Ad5CMV (empty vector), trigger apoptosis in these extremely resistant cell types is a topic ␤ Ad5CMVLacZ ( -galactosidase), and Ad5CMVE2F-1 of intense investigation. (E2F-1) at multiplicity of infection of 100. Western blot The process of apoptotic cell death is very distinct and analysis confirmed marked overexpression of E2F-1 in both involves unique morphological alterations, which include cell cell lines. By 5 days after infection, E2F-1 overexpression shrinkage, plasma and nuclear membrane blebbing, chromatin resulted in >25-fold reduction in cell growth and >90% loss condensation, and the formation of apoptotic bodies (7). There of cell viability in both cell lines. Cell cycle analysis of are two distinct phases during apoptotic cell death: commitment

Ad-E2F-1-infected cells revealed an increase in G2/M and and execution. The Bcl-2 family members appear to regulate the

sub-G1 populations. By in situ terminal deoxynucleotidyl cellular commitment to survive or die when challenged with transferase (Tdt)-mediated nick end labeling analysis, evi- various apoptotic stimuli (8, 9). The execution phase of apo- dence of apoptosis was observed including internucleosomal ptosis is controlled by a sequential cascade of activation by DNA fragmentation and the formation of apoptotic bodies. proteolytic cleavage of the caspase family of proteins (10–12). In addition, caspase-3 and poly(ADP-ribose) polymerase ap- Apoptotic cell death is induced by a number of stimuli. DNA- optotic fragments were detected by 48 h after treatment with damaging treatments such as UV irradiation and a variety of Ad-E2F-1. Of mechanistic importance, overexpression of chemotherapeutic drugs have been shown to induce apoptosis, which can involve both p53-dependent and -independent mech- E2F-1 caused a G2/M arrest followed by increased levels of c-Myc and p14ARF proteins. Additionally, expression of the anisms (13–15). antiapoptotic Bcl-2 family member Mcl-1 was down-regu- Recent evidence points to an alternative apoptotic pathway that results in the induction of the tumor suppressor protein lated in E2F-1-overexpressing cells. In conclusion, E2F-1 ARF ARF overexpression initiates apoptosis and suppresses growth in p14 . p14 is produced from the transactivation of an alternate reading frame found in the ink4a tumor suppressor HT-29 and SW620 colon adenocarcinoma cells. Overexpres- gene (16, 17). This novel apoptotic pathway involving p14ARF sion of E2F-1 triggers apoptosis and is associated with up- is stimulated under hyperproliferative or oncogenic conditions regulation of c-Myc and p14ARF proteins and down-regula- but is not stimulated after DNA damage (18, 19). Therefore, p14ARF-associated apoptosis is believed to constitute a separate pathway to programmed cell death. Paradoxically, the same oncogenes and growth factors that can induce cellular prolifer- Received 10/3/00; revised 8/17/01; accepted 8/17/01. ation can also cause apoptosis and growth arrest. To date, The costs of publication of this article were defrayed in part by the overexpression of the transcription factors E2F-1 and c-Myc as payment of page charges. This article must therefore be hereby marked well as activated oncogenic Ras (H-Ras) and an adenoviral advertisement in accordance with 18 U.S.C. Section 1734 solely to protein, E1a, have been shown to induce increased expression of indicate this fact. ARF 1 Supported by American Cancer Society Award 96-55, Alliant Com- p14 followed by either grow arrest and/or apoptosis (20– munity Trust Fund Award 96-46, The Mary and Mason Rudd Founda- 23). However, thus far, only c-Myc-like E box motifs and a tion Award, and the Center for Advanced Surgical Technologies of potential E2F-1 binding site (GCGGGAAA) have been identi- Norton Hospital. fied in the specific promoter for p14ARF (20, 23). 2 To whom requests for reprints should be addressed, at University of Louisville-Brown Cancer Center, 529 South Jackson Street, Louisville, E2F-1 has been shown to possess both oncogenic and KY 40202. Phone: (502) 852-5211; Fax: (502) 629-3393; E-mail: apoptotic properties. As a transcription factor, E2F-1 is immi- [email protected]. nently involved in the regulation of the cell cycle. When un-

bound to the pRb3, E2F-1 transactivates genes required for DNA scribed previously (29, 36). The adenoviruses Ad5 and Ad- synthesis and progression into S phase. Early studies with E2F-1 E2F-1 were generous gifts from Ta Jen Liu (University of suggested that this protein can function as an oncogene when Texas, MD Anderson Cancer Center, Houston, TX). Ad-LacZ overexpressed (24–26). However, increasing E2F-1 activity by was a generous gift from Dr. Brent French (University of overexpression of E2F-1 has been shown to lead to apoptosis in Virginia, Charlottesville, VA). For preparation of large virus many cell types (27–31). When E2F-1 is overexpressed, an stocks, 293 cells were infected at MOI 1–5 and harvested after unscheduled S phase entry occurs and is often followed by a cytopathic effect became visible (24–48 h). Cells were har- ϫ G2/M arrest and apoptosis (27, 28, 32). Furthermore, recent vested and lysed in 1 PBS containing 1% sucrose, and virus studies demonstrate that the apoptotic function of E2F-1 is aliquots were stored at Ϫ70°C. Titers were determined by either independent of its transcriptional function but is dependent on plaque assay on 293 cells for plaque forming units/ml or spec- its DNA-binding capability (33, 34). In fact, E2F-1 has been trometer reading at 260 nm for particles/ml (37) Ad5 (empty linked to two very distinct apoptotic pathways: (a) E2F-1 pro- vector) and Ad-LacZ (expressing nuclear-localized ␤-gal) were tein levels increase in a variety of cell lines after DNA damage- used as control vectors. induced apoptosis and induction occurs independent of pRb or Virus Infection. For infections, 2.5 ϫ 105 cells were p53 status (35); and (b) overexpression of E2F-1 has been plated in 75-mm tissue culture flasks and allowed to incubate for shown to result in the up-regulation of p14ARF and apoptosis in 48 h. Cells were then infected by adding the adenoviral vectors both p53-wild-type and p53-null cells (20). Still, to date, little is in 1800 ␮l of McCoys 5A medium at MOI of 100 plaque known of the mechanism of E2F-1-mediated apoptosis. forming units/cell. Mock infection was performed by treatment In the present study, we investigate the effect of adenovi- of cells with vehicle (medium) only. After a 2-h incubation at ral-mediated gene transfer of E2F-1 on colon cancer cell growth. 37°C, 28.2 ml of fresh McCoys 5A medium with 5% FBS was The results of this study demonstrate that adenoviral-mediated added. Cells were trypsinized at 24, 48, 72, and 120-h time gene transfer is an efficient method for the delivery of genes to points, counted via a Coulter counter, and prepared for various colon cancer cells, because E2F-1 was highly expressed after testing as outlined below. Optimal concentrations of viral vec- infection. The overexpression of E2F-1 induced the cells to tors causing expression in 80–100% of the cells without visible ␤ accumulate in G2-M within 24 h after treatment with apoptotic toxic effect were determined by infection of cells with the -gal DNA fragmentation in Ͼ50% of the cells by 120 h after treat- expressing virus at different doses and subsequent staining with ment. Mechanistically, we found that p14ARF and c-Myc were X-gal (French). up-regulated, and Mcl-1 expression was abrogated after E2F-1 ␤-Gal Assay. The cell lines infected with Ad-LacZ were gene transfer. These results suggest that p14ARF, c-Myc, and assayed for ␤-gal expression by the X-gal staining method as Mcl-1 play an important role in E2F-1-mediated apoptosis. described previously (38). Briefly, 48 h after infection, the tumor cells were washed with PBS and fixed in 2% (vol/vol) MATERIALS AND METHODS formaldehyde and 0.2% (vol/vol) glutaraldehyde in PBS (pH Cell Lines and Culture Conditions. Human colon can- 7.4) for 5 min at 4°C. The cells were then washed and stained cer cell lines HT-29 and SW620 (both mutant p53) were pur- with X-gal solution [1 mg/ml 5-bromo-4chloro-3-indolyl-b- chased from American Type Culture Collection (Rockville, galactopyranoside, 5 mM K4Fe(CN)6,5mM K3Fe(CN)6,2mM MD). Both cell lines were cultured in McCoy’s 5A medium MgCl2 in PBS (pH 6.5)] for 12–18hat37°C. Blue staining of modified and supplemented with 10% heat-inactivated FBS, cell nuclei identified transduced cells. Mock-infected cells and streptomycin/penicillin (100 units/ml). The transformed embry- cells transduced with other adenoviral vectors served as con- onic kidney cell line 293 was grown in minimum essential trols. ␣-medium [medium with high glucose (4.6 g/l)], supplemented Immunoblotting Procedures. Cells were harvested at with 10% FBS and 1% antibiotic/antimycotic. All of the cell selected time points and lysed in RIPA lysis buffer (50 mM culture reagents were obtained from Life Technologies, Inc. trisect, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) or in PBS containing 1% NP40. A protease (Bethesda, MD). Cells were cultured in a 5% CO2 incubator at 37°C, and the medium was changed every 3 days. inhibitor cocktail [4-(2-aminoethyl)-benzenesulfonyl fluoride, Preparation of Adenoviral Vectors. A replication- pepstatin A, transepoxysuccinyl-L-leucylamido(4-guanidino)bu- defective recombinant adenoviral vector (Ad-5) that has had tane(E-64), bestatin, leupeptin, and aprotinin] was added to each portions of the early region 1 deleted, and a constitutive CMV lysis buffer. Protein concentration was determined by BIO-RAD promoter inserted was used in these experiments. In addition to DC protein assay (BIO-RAD, Hercules, CA). For the detection ␮ the empty vector (Ad5), two vectors containing either the trans- of Bcl-2, Bax, Bak, Bcl-x, Mcl-1, and caspase-3 proteins, 30 g gene E2F-1 or LacZ were used. These vectors have been de- of protein were loaded in each lane of a 12% SDS-Page gel. A 10% SDS-Page gel was used for the detection of c-Myc, PARP, and E2F-1 and a 15% SDS-Page gel for the detection of p14ARF. Protein was transferred to a polyvinylidene difluoride membrane (Bio-Rad). Proteins were detected using antihuman Bcl-2, 3 The abbreviations used are: pRb, retinoblastoma gene product; FBS, Bax, Bak, Bcl-x, Mcl-1, c-Myc (PharMingen), E2F-1, p14ARF fetal bovine serum; Ad5, Ad5CMV; Ad-E2F-1, AdCMVE2F-1; Ad- (Santa Cruz Biotechnology), PARP (Calbiochem), and ␤ LacZ, Ad5/CMV/nls/LacZ; MOI, multiplicity of infection; -gal, ␣ ␤galactosidase; RIPA, radioimmunoprecipitation assay; PARP, poly- Caspase-3 (Transduction Laboratories, Lexington, KY). - (ADP-ribose) polymerase; TUNEL, terminal deoxynucleotidyl transfer- Actin polyclonal antibody (Sigma Chemical Co., St. Louis, MO) ase (Tdt)-mediated nick end labeling. was used as control to demonstrate equal loading and transfer.

Enhanced chemiluminescence reagents were used to detect the signals according to the manufacturer’s instructions (Amer- sham, Arlington Heights, IL). Intensity of each protein band was quantified using NIH image software after scanning of the image. Cell Growth and Viability Assays. Cells were harvested at indicated time points, and cell viability was estimated as the proportion of cells that excluded 0.2% trypan blue. Cell growth (suspension/ml) was estimated by counting 100 ␮lof cells with a Coulter counter ZM at each time point. Cell Cycle Analysis. Adherent and nonadherent cells Fig. 1 Overexpression of E2F-1 in colon cancer cell lines. HT-29 and were harvested, washed once with PBS, fixed in 70% (vol/vol) SW620 were treated by mock infection, or infection with Ad-5 (empty ethanol, and stored at 4°C for up to 1 week. Cells were pelleted, vector), Ad-LacZ (LacZ), or Ad-E2F-1 (E2F-1). E2F-1 expression was assessed 24, 48, 72, and 120-h later by Western blot analysis. Blot washed once with phosphate buffered saline, and resuspended in represents cells harvested at the 72 h time point and lysed in RIPA lysis propidium iodide solution [50 ␮g/ml PI and 0.5mg/ml in PBS buffer. Blot is representative of at least two separate experiments. (pH 7.4)] for 30 min in the dark. Cells were analyzed for DNA ␣-Actin was used as a control to demonstrate an equal loading and content by fluorescence-activated cell sorting (FACScan; Bec- transfer. ton Dickinson). Data were analyzed using a CellFit Cell-Cycle Analysis program (version 2.01.2). The subdiploid population was calculated as an estimate of the apoptotic cell population. Overexpression of E2F-1. Western blot analysis con- The proportion of G ,S,G-M and subG phase cells was 1 2 1 firmed marked overexpression of the E2F-1 transgene after estimated each as a percentage of the total (10,000) events. infection with the Ad-E2F-1 vector in both the HT-29 and Confirmation of Apoptosis. Several methods were used SW620 colon cancer cell lines (Fig. 1). The presence of multiple to confirm apoptotic cell death. First, simultaneous evaluation of bands of E2F-1 likely indicates various species of hyper- and morphological and biochemical changes attributable to apopto- hypophosphorylated E2F-1 as described previously (40). In sis was performed via in situ TUNEL. The TUNEL assay contrast, baseline expression of E2F-1 in mock-infected, Ad-5, identifies internucleosomal DNA strand breaks as well as mor- or Ad-LacZ-infected control cells was barely detected. phological changes characteristic of apoptosis. Cells were fixed Effects of Adenoviral Vector-mediated E2F-1 Expres- in 4% formaldehyde in PBS (pH 7.4) for 15 min at room sion on Cell Growth. At 24 h after infection, there was no temperature. After centrifugation, cells were resuspended in apparent growth inhibition in E2F-1-overexpressing cells com- 80% ethanol and stored at 4°C for up to 1 week. A TdT-FragEL pared with mock-infected, Ad5, or Ad-LacZ-infected cells (Fig. DNA fragmentation detection kit (Calbiochem, Oncogene Re- 2, A and B). By 48 h, however, cells infected with the Ad-E2F-1 search Products, Cambridge, MA) was used to detect apoptosis vector exhibited marked growth inhibition, which was even according to instructions provided by the manufacturer. The more pronounced at 72 h and 120 h. The effects were similar in detection of caspase 3 and PARP cleavage has been shown to be both HT-29 and SW620 (both mutant p53) cell lines. A dramatic a sensitive method for confirmation of apoptosis (39). Cleavage decline in cell viability was also detected in the E2F-1-overex- assays were performed using a monoclonal mouse anti-PARP pressing cells, as assessed by trypan blue exclusion (Fig. 3, A antibody (Calbiochem, Oncogene Research Products, Cam- and B). These data indicate that overexpression of E2F-1 effec- bridge, MA) at a dilution of 1:100 and anti-Caspase-3 (Trans- tively inhibits cell growth and induces cell death in colon duction Laboratories, Lexington, KY) at a dilution of 1:1000. cancer. Statistics. Unless stated otherwise, numerical data repre- Effects of Ad-E2F-1 Infection on Cell Cycle. To addi- sents mean values for at least two experiments. SE, unless tionally investigate the mechanism of E2F-1-induced colon can- shown, were Ͻ10% of the value of the respective data point. cer cell death, cell cycle distribution after the Ad-E2F-1 was Student’s t test was used to evaluate whether differences be- analyzed. As shown in Fig. 4 and Tables 1, a and b, E2F-1 Ͻ tween data sets were statistically significant. Only Ps 0.05 overexpression was associated with a decrease in G1 phase cells were considered significant. and a concomitant increase in sub-G1 and G2-M populations by 24 h in both cell lines. By 5 days after infection, the subdiploid population predominated. Mock-infected, Ad5, and Ad-LacZ- RESULTS infected cells showed little alteration of normal cell cycle pro- Transduction Efficiency of Adenoviral Vectors. To es- files and few subdiploid cells in both cell lines. Quantitation of timate the transduction efficiency of adenoviral vectors, the two cell cycle data are shown in Table 1, a and b. These data suggest human colon cell lines were infected with Ad-LacZ at MOI that E2F-1 overexpression is associated with premature S phase Ն Ͼ ranging from 25–150. At MOI of 75, 85% of cells demon- entry, G2-M arrest, and DNA fragmentation in HT-29 and strated nuclear staining for ␤-gal. Increased intensity of ␤-gal SW620 colon cancer cells. staining was demonstrated with increased MOI up to 150 (data E2F-1-mediated Cell Death Is Attributable to Apopto- not shown). No significant cytotoxicity was observed with MOI sis. In addition to internucleosomal DNA fragmentation and up to 150. All of the subsequent experiments were performed at the cellular morphological markers seen during apoptosis, a MOI of 100. cascade of molecular events can serve as early biochemical

Fig. 2 The effect of Ad-E2F-1 on colon cancer cell growth. A, HT-29 cells; B, SW620 cells. Cell growth was determined by cell counting with Fig. 3 Loss of cell viability after infection with Ad-E2F-1. A, HT-29 a Coulter counter ZM. Results are expressed as cells/ml at time points cells; B, SW620 cells. At 1, 3, and 5 days after mock infection, or 24, 72, and 120 h after infection. Each data point represents the mean Ϯ infection with Ad-5 (empty vector), Ad-LacZ (LacZ), or Ad-E2F-1 SE of three separate experiments. SEs are shown only if Ͼ10% of the (E2F-1), both floating and adherent cells were harvested, counted, and respective value. scored for viability by the trypan blue exclusion method. Results are expressed as the percentage of viable cells (live cells/total cells) and represent the mean of three independent experiments. SEs are shown only if Ͼ10% of the respective viability value. markers, including activation of the caspase family of proteins and PARP cleavage. Cleavage of the nuclear enzyme PARP by caspase-3 has been proposed as one of the earliest events in the execution phase of apoptosis (9, 10). On activation, caspase-3 is control the cellular commitment to programmed cell death, cleaved forming a Mr 17,000 active fragment (41, 42). immunoblots were performed to detect any measurable changes Caspase-3 preferentially cleaves on the carboxyl side of the in the levels of Bak, Bax, Bcl-xl, Bcl-xs, Bcl-2, and Mcl-1 (Fig. tetrapeptide sequence DEVD, a sequence present in PARP (42). 6, A and B). Results confirmed that no changes in intracellular

Proteolysis of the Mr 113,000 PARP protein by caspase-3 gen- levels of Bax, Bak, or Bcl-xl (Bcl-xs and Bcl-2 were not erates 89 and Mr 24,000 cleavage products (39). Cleavage of detected in either cell line) occurred by 3 days after infection in caspase-3 and PARP that is consistent with apoptosis was de- any of the samples. However, expression of the antiapoptotic tected in the E2F-1-overexpressing cells (Fig. 5, A and B)by protein Mcl-1 disappeared in the cells infected with E2F-1 in 48 h. Additionally, in the HT-29 cell line, low levels of both HT-29 and SW620 cells by 72 h after infection. These caspase-3 are evident in mock- and LacZ-infected cells (data not results suggest that the cleavage or down-regulation of Mcl-1 shown; Fig. 5A); however, after infection with E2F-1, levels of might be a controlling factor in E2F-1-induced apoptosis. Cleav- caspase-3 increase dramatically. It is unclear why this induction age of the proapoptotic protein, Bax, was also detected in the of caspase-3 protein occurs in HT-29 cells. Mock-infected, Ad5, E2F-1-overexpressing cells, which demonstrates the activation or Ad-LacZ-infected cells demonstrated none of these morpho- of calpains and caspases (43, 44). However, a number of studies logical or biochemical changes. Therefore, these data confirm show that inhibition of calpains inhibits Bax cleavage but does the involvement of the caspase cascade in E2F-1-induced apo- not inhibit PARP cleavage or apoptotic death (43, 44). There- ptosis of these cell lines. fore, cleavage of Bax is not expected to be involved in the cellular Using in situ TUNEL assay, it is possible to simultaneously commitment to apoptosis but rather a result of apoptosis. observe morphological changes and DNA fragmentation con- Intracellular Levels of c-Myc and p14ARF. Because sistent with apoptosis. Five days after infection with Ad-E2F-1, overexpression of E2F-1 is known to result in unscheduled S apoptotic evidence was observed including internucleosomal phase entry and, therefore, a potential hyperproliferative signal, DNA fragmentation and the formation of apoptotic bodies by we suspected that the p14ARF apoptotic pathway might be TUNEL assay in the HT-29 and SW620 cells (data not shown). involved. As shown in Fig. 7, up-regulation of p14ARF was Intracellular Levels of Bcl-2 Family Members. Be- detected in both cell lines 72 h after infection with Ad-E2F-1. cause the Bcl-2 family of proteins are believed to interact to To additionally characterize the mechanisms underlying E2F-1-

Fig. 4 Cell cycle analysis. Ad-E2F-1 infection resulted in a decrease in

G1 phase and a concomitant increase in G2-M and sub-G1 populations by 1 day after treatment in HT-29 and SW620 cells. A pronounced subdiploid peak was evident at 120 h after treatment in both cell lines.

Table 1 Cell cycle distribution after E2F-1 adenovirus treatment. a. HT-29 Time point/phase Mock Ad-5 Ad-LacZ Ad-E2F-1 Fig. 5 Activation of the caspase cascade in Ad-E2F-1-infected cells. A,

24 h: SubG1 2.8 3.1 3.4 18.5 expression of precaspase-3 and the active Mr 17,000 fragment, and G1 44.7 45.7 45.3 25.0 PARP Mr 85–90,000 apoptosis-related cleavage fragment observed in A S 32.5 34.9 33.4 25.2 (HT-29 cells) and B (SW-620) cells 24, 48, and 72 h after treatment with ␣ G2/M 20.1 16.6 17.9 31.3 Ad-LacZ and Ad-E2F-1. A and B, -actin was used as internal control 72 h: SubG1 2.1 7.6 3.8 49.8 to demonstrate equal loading of lanes. Cells were lysed in RIPA lysis G1 70.8 61.8 69.3 26.0 buffer for the detection of caspase-3 and PARP. Equivalent results were S 10.0 17.0 11.7 16.2 obtained in three independent experiments. G2/M 17.1 13.6 15.2 8.0 120 h: SubG1 2.2 5.2 3.0 63.1 G1 81.1 81.7 85.1 21.3 S 2.6 3.8 6.0 12.7

G2/M 14.0 9.4 5.8 2.9 was detected only after c-Myc was induced, and a large majority b. SW620 of the cells were apoptotic. The protein expression timeline seen in these results suggests that E2F-1 may induce up-regulation of Time point/phase Mock Ad-5 Ad-LacZ Ad-E2F-1 p14ARF with the cooperation of c-Myc in these cell lines.

24 h: SubG1 3.7 4.7 3.6 13.7 G 51.0 44.4 39.1 19.1 1 DISCUSSION S 14.6 13.7 22.4 22.4

G2/M 30.6 37.2 34.9 44.7 In the present study, we used an adenoviral vector system 72 h: SubG1 3.2 8.5 8.8 47.1 to transfer the E2F-1 gene to colon cancer cells and analyzed the G1 56.9 35.1 48.4 16.0 ability of E2F-1 to trigger apoptosis. Here, for the first time, we S 12.5 13.9 14.5 13.7 demonstrate that treatment of colon cancer cells with adenovi- G /M 27.1 33.5 28.2 23.2 2 ruses expressing the E2F-1 gene can effectively induce apopto- 120 h: SubG1 1.6 8.6 8.9 52.2 G1 62.3 52.8 58.7 19.6 sis. Mechanistically, our data supports previous studies per- S 15.0 12.2 9.5 17.7 formed in other cell types in that overexpression of E2F-1 G2/M 21.1 26.4 22.9 10.5 induces G2-M arrest and cell death (27–31). However, our report contributes the additional understanding that E2F-1- mediated apoptosis involves increased expression of p14ARF and c-Myc and down-regulation of Mcl-1. This data indicates mediated apoptosis, we probed for c-Myc, which has been that p14ARF, c-Myc, and Mcl-1 may play an important role in shown to up-regulate p14ARF when overexpressed and induce E2F-1-induced apoptosis in colon cancer. apoptosis under hyperproliferative conditions (22). A dramatic A growing body of evidence indicates that oncogenes and increase in c-Myc protein levels was observed in the E2F-1- growth factors can induce proliferation and promote cellular overexpressing cells by 48 h after infection in both cell lines survival but when overexpressed can cause apoptosis and (Fig. 7). In the E2F-1-overexpressing cells, p14ARF expression growth arrest (18, 45, 46). This mechanistic duality appears to

Fig. 7 Induction of p14ARF and c-Myc. A, HT-29; B, SW620 cells harvested and lysed in NP40 (p14ARF) or RIPA (c-Myc) lysis buffer at 24, 48, and 72 h after infection for immunoblot analysis. Analysis performed as described in “Materials and Methods.” Blots representa- ␣ Fig. 6 Intracellular changes in Bcl-2 family members. Immunoblot tive of at least two separate experiments. -Actin was used as internal analysis was performed as described in “Materials and Methods.” Blots control to demonstrate equal loading of lanes. representative of at least three separate experiments. Cells harvested and lysed in NP40 lysis buffer for immunoblot analysis at 72 h after treatment. ␣-Actin was used as internal control to demonstrate equal loading of lanes. always required (27–29, 33, 55). Although many studies show ARF that a p14 -induced G1 arrest and subsequent apoptotic response is p53 dependent, a separate study showed that overex- be true for E2F-1. Early studies with E2F-1 suggested that this pression of E2F-1 in p53-null Saos-2 cells resulted in acceler- protein can function as an oncogene when overexpressed be- ated entry into DNA synthesis, a dramatic increase in p14ARF, cause of the transactivation of a series of genes, which facilitate and apoptotic cell death (20). Interestingly, p14ARF has been transition of the cell from G1 to S phase (24, 26, 31, 47–49). On shown to be dispensable for cell proliferation and, therefore, is the other hand, in vivo experiments using homozygous e2f-1 null believed to have a specific role for priming or mediating cell mice demonstrate increased cell proliferation and neoplasia in death (22). In the present study, two colon cancer cell lines were several tissues, thus, supporting the putative tumor suppressor examined: HT-29 and SW-620, both of which posses mutant activity of E2F-1 (50). Furthermore, overexpression of E2F-1 p53. Overexpression of E2F-1 resulted in accumulation of cells ARF has been shown to induce apoptosis in several cell types, indi- in G2-M, increased levels of p14 , and apoptosis. Thus, cating that E2F-1 plays a role not only in stimulating cellular although wild-type p53 may be required for p14ARF-associated ARF proliferation but in coordinating programmed cell death G1 arrest, it appears that E2F-1/p14 -associated G2-M arrest (27–31). and apoptosis can proceed without regard to the status of p53 in Recent evidence indicates that E2F-1 overexpression can these cell lines. induce an alternate apoptotic pathway that involves increased Like E2F-1, the transcription factor c-Myc can promote expression of p14ARF, a protein with tumor suppressor activity both cell survival and death and has been shown to have sepa- (20, 32). The p14ARF-associated pathway is found to be stimu- rable regulatory functions in both cell proliferation and death. ARF lated by hyperproliferative signals and can result in a G1 or Interestingly, p19 has been shown to be involved in c-Myc- G2-M cell cycle arrest and apoptosis (51). A substantial amount induced apoptosis (22) and has been identified as a c-Myc target ARF of data indicates that p14 facilitates a G1 arrest by neutral- gene (23). A relationship between E2F-1 and c-Myc is indicated izing MDM-2 activity thereby causing stabilization, accumula- with data that show E2F transcription factors up-regulate c-Myc, tion, and activation of p53 and ultimate transactivation of p21 and pRb-sequestered E2F induces the down-regulation of c-Myc (17, 51–53). However, virtually no data exists regarding the (56, 57). Moreover, DNA-damaging treatments with etoposide ARF function of p14 during a G2-M arrest. In the current study of and adriamycin resulted in the up-regulation of E2F-1 followed colon cancer, overexpression of E2F-1 resulted in G2-M arrest by an increase in c-Myc and apoptosis in medulloblastoma and and apoptosis. As shown in Fig. 7, overexpression of E2F-1 in glioma cancer cell lines (35). Given this data regarding the HT-29 and SW620 cells was followed by an increase in the level E2F-1/c-Myc relationship and the fact that p19ARF was identi- of p14ARF. These findings indicate that apoptosis induced by the fied as a target gene of c-Myc, we were curious to see if c-Myc overexpression of E2F-1 is associated with increased levels of was induced after overexpression of E2F-1 in our cells. Fig. 7 p14ARF in colon cancer cells. demonstrates that up-regulation of c-Myc is detected by 48 h There is evidence that overexpression of E2F-1 induces the after infection with Ad-E2F-1 and an increase in p14ARF by the accumulation of p53, implicating p53 in E2F-1-mediated apo- 72 h time point. These findings indicate that overexpression of ptosis (54). An increase in p14ARF expression may explain the E2F-1 may not directly result in an increase in p14ARF but may accumulation of p53 seen after overexpression of E2F-1 in some require the cooperation of c-Myc expression in these cell lines. wild-type p53 cell lines. Whereas it was originally believed that The fact that c-Myc expression was elevated in the E2F- p53 was essential for E2F-1-mediated apoptosis, the results 1-overexpressing cells does suggest that c-Myc may be involved from a variety of experiments make it clear that active p53 is not in E2F-1-mediated apoptosis in colon cancer. Substantial evi-

dence indicates that c-Myc and E2F-1 share specific apoptotic 2. Bedi, A., Pasricha, P. J., Akhtar, A. J., Barber, J. P., Bedi, G. C., pathways involving p14ARF, but also take distinct routes to Giardiello, F. M., Zehnbauer, B. A., Hamilton, S. R., and Jones, R. J. induce cell death (18, 23, 58). Recent studies using centrifugal Inhibition of apoptosis during development of colorectal cancer. Cancer Res., 55: 1811–1816, 1995. elutriation demonstrated that high ectopic levels of E2F-1 in 3. Vogelstein, B., Fearon, E. R., Hamilton, S. R., Kern, S. E., Preis- Rat1 cells induced apoptosis specifically from cells in G , but 1 inger, A. C., Leppert, M., Nakamura, Y., White, R., Smits, A. M. M., overexpression of c-Myc triggered apoptosis from cells in G1 and Bos, J. L. Genetic alterations during colorectal-tumor development. and G2-M phases (58). Interestingly, Rat1 cells accumulated in N. Engl. J. Med., 319: 525–532, 1988. G2-M after overexpression of either oncogene; however, only 4. 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