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[Cancer Research 63, 6229-6236, October 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Adenovirus Type 5 E1A Sensitizes Hepatocellular Carcinoma Cells to Gemcitabine1

Wei-Ping Lee2, Dar-In Tai, Sun-Lung Tsai, Chau-Ting Yeh, Yee Chao, Shou-Dong Lee and Mien-Chie Hung

Department of Internal Medicine [W-P. L., S-D. L.] and Cancer Center [Y. C.], Taipei Veterans General Hospital, Taipei, Taiwan; The Liver Research Unit, Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Taoyuan, Taiwan [D-I. T., S-L. T., C-T. Y.]; Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [M-C. H.]; and Department of Biochemistry, Yang-Ming University, Taipei, Taiwan [W-P. L.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocellular carcinoma (HCC) is resistant to conventional chemotherapy. A few clinical trials have shown that the cytidine analogue gemcitabine appears to have antitumor activity for HCC, but the overall survival times remain to be improved. In this study, we examined the synergistic effect of adenovirus type 5 E1A (E1A) and gemcitabine on HCC and found that E1A sensitized J5, J7, Huh7, and HepG2 HCC cells to gemcitabine. To further study the E1A-mediated chemosensitization, we established stable cell lines that expressed the E1A gene and then examined whether E1A could have proapoptotic activity while expressed in HCC cells. Our results clearly showed that E1A sensitized HCC cells to gemcitabine through induction of apoptosis. To study the underlying mechanism, we tested nuclear factor (NF)-{kappa}B activity and found that NF-{kappa}B was activated in HCC cells treated with gemcitabine but not in HCC cells that expressed E1A. Occurrence of apoptosis entails cleavage of poly (ADP-ribose) polymerase (PARP), a nuclear protein involved in DNA repair, genome stability, and maintenance of telomere length. Our study showed that gemcitabine enhanced PARP expression. However, E1A did not induce PARP cleavage but rather suppressed PARP expression at the transcriptional level. Further study showed that both NF-{kappa}B and PARP played protective roles in the prevention of E1A+gemcitabine-induced apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HCC3 is a major cancer in the world and among the leading causes of cancer death in Asian countries. Chronic infection of either hepatitis B or hepatitis C, alcohol consumption, and aflatoxin ingestion are known risk factors (1, 2, 3, 4, 5) . The current choices for HCC treatment include surgical resection, percutaneous ethanol injection, chemoembolization, and local or systemic chemotherapy. Among these treatments, the chance of achieving disease-free state for HCC remains lower than those for other types of cancer. Surgical resection may provide an occasional incidence of cure; however, it can be performed only in selected patients whose tumors are small and away from major vessels and have not metastasized to extrahepatic organs. In general, patients with unresectable HCC have a dismal prognosis, and actually, these patients do not benefit much from nonsurgical treatments, such as regional chemotherapy and chemoembolization (6, 7, 8, 9, 10) . Thus, the search for more effective chemotherapeutic agents is still ongoing, and new regimens are under active investigation.

The cytidine analogue gemcitabine dFdC has been used to treat solid tumors. Promising results have been reported for the treatment of colorectal, breast, pancreatic, renal, and lung cancer (11, 12, 13, 14) . Gemcitabine requires nucleoside transporters in the plasma membrane to enter cells (15) . Gemcitabine (dFdC) is phosphorylated by deoxycytidine kinase to dFdCMP, dFdCDP, and dFdCTP. dFdCTP is incorporated into DNA and thereby blocks DNA replication and repair (16) . dFdCTP can also be incorporated into RNA and can inhibit CTP synthetase (17) . dFdCMP inhibits dCMP deaminase (18) . dFdCDP inhibits ribonucleotide reductase, which is essential for generation of deoxyribonucleoside triphosphates required for the synthesis of DNA, leading to depletion of the DNA precursor pool, deoxynucleoside triphosphate (19 , 20) . These actions will result in accumulation of dFdCMP, dFdCDP, and dFdCTP because dFdCMP is not degraded within cells, and a reduction of dCTP will reduce feedback inhibition of deoxycytidine kinase (19) . Furthermore, inhibition of CTP synthesis will also enhance incorporation of dFdCTP into RNA (19) . At present, systemic chemotherapy has limited value in the treatment of HCC. Recently, gemcitabine has been put on clinical trials to treat hepatocellualr carcinoma. The reported median survival time was 18.7 weeks by Yang et al. (21) , 20 weeks by Kubicka et al. (22) , 27 weeks by Fuchs et al. (23) , and 34 weeks by Ulrich-Pur et al. (24) . It appears that gemcitabine monotherapy has not given an encouraging treatment result.

The type 5 adenovirus has been wildly used in delivering genes into mammalian cells. E1A is one of the early viral genes, which codes for two major proteins of 243 and 289 amino acids by alternative splicing in two exons. The early viral proteins can activate or repress transcription of several viral or cellular genes and thereby regulate the cell cycle (25) . E1A was first appreciated for its ability to inhibit Her-2/neu expression in both rodent and human cancer cells. The human Her-2/neu oncogene is overexpressed in many human cancers, and E1A has been shown to act as a tumor suppressor by down-regulating Her-2/neu transcription (26, 27, 28, 29) . In a number of studies, E1A was shown to reduce tumor growth in cancer cells that do not overexpress Her-2/neu (30, 31, 32, 33) . The expression of E1A reduced the anchorage-independent growth and tumorigenecity in a number of malignant cell lines, including ovarian cancer, sarcoma, and lung cancer (30, 31, 32, 33) . E1A has shown promising therapeutic effects in a Phase I trial for breast cancer (34) and a Phase I trial for head and neck cancer (35) . Animal studies of E1A-mediated gene therapy for HCC have also shown significant tumor-suppressing effect (36 , 37) . It appears that E1A could be a universal tumor suppressor in a variety of tumors. In addition to the tumor-suppressing activity, E1A gene transfer has resulted in increased cell sensitivity to paclitaxel (Taxol) in human breast cancer (38) , Adriamycin in colon and HCC (39 , 40) , and cisplatin and etopside in sarcoma cells (41) .

In the current study, we demonstrate that E1A suppresses growth of J5, J7, Huh7, and HepG2 HCC cell lines and sensitizes these cells to gemcitabine. In addition, we found that E1A acted as a proapoptotic molecule that induced apoptosis while E1A-expressing J5 cells were exposed to gemcitabine. Our previous study showed that E1A inhibited NF-{kappa}B activation in tumor cells treated with {gamma}-irradiation or TNF-{alpha} (42 , 43) . The current study gives additional evidence to support our previous finding that suppression of NF-{kappa}B activity plays a role in E1A-mediated proapoptotic activity. To further study E1A-mediated chemosensitization, we analyzed the expression of PARP and found that instead of inducing cleavage of PARP, E1A suppressed the expression of PARP at the transcriptional level in HCC cells treated with gemcitabine. PARP plays an important role in DNA repair. Because gemcitabine can be incorporated into DNA, resulting in chain termination during DNA replication, suppression of PARP by E1A provides a mechanism by which E1A sensitizes HCC cells to gemcitabine.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Line and Cell Culture.
J5 and J7 are human HCC cell lines (44 , 45) . Huh7 and HepG2 are well-known human HCC cell lines. Cells were grown in Dulbecco’s modified Eagle’s + F12 (1:1) medium (Life Technologies, Inc.) supplemented with 10% FCS in a humidified atmosphere of 5% CO2 at 37°C.

Adenoviral Vectors.
Recombinant, replication-deficient adenoviral vectors were prepared by superinfection of HEK293 cells. Viruses were purified and titered by a standard protocol as described previously (46) . Two adenoviruses were used: (a) Ad.E1A(+) is an adenovirus with E1B and E3 deletions; and (b) AdE1A(-) is an adenovirus with E1A and E3 deletions (47) .

Establishment of Stable Cell Lines.
The E1A cDNA was cloned into a pSVneo vector carrying the aminoglycoside phosphotransferase gene (APH). Expression of the E1A gene was driven by the E1A gene’s promoter and that of APH by the SV40 promoter. The E1A gene transfection was carried out using lipofectin (Invitrogen, Inc.). Briefly, 6 µg of pSVneo-E1A plasmid along with the lipofectin mixture were transfected into 1 x 106 J5 HCC cells cultured in a 6-cm tissue culture dish. Approximately 8 h after transfection, the cells were washed with PBS, cultured in fresh medium for 24 h, and then split 1:20. Then, the cells were grown in a selection medium containing 500 µg/ml G418 (Bio101, Inc.) for 3–5 weeks, after which, individual G418-resistant clones were picked out and expanded to mass culture.

In Vitro Cytotoxicity Assays by Trypan Blue Staining.
Exponentially growing cells were harvested by trypsin digestion, washed with PBS, and resuspended in appropriate amounts of medium. Cells (2 x 105) were plated onto 6-cm tissue culture dishes, to which was added 1 x 106 plaque-forming unit AdE1A(-) or AdE1A(+) virus in replicates of three. Twelve hours after addition of a virus, cells were treated with different concentrations of gemcitabine, incubated at 37°C for 72 h, washed twice with PBS, and then digested with PBS. Viable cells were counted with a microscope. Dead cells were excluded by trypan blue staining. To study E1A-mediated chemosensitization in E1A stable transfectant, cells (J5 control and E1A transfectants, 2 x 105) were plated onto 6-cm tissue culture dishes overnight and then treated with different concentrations of gemcitabine. Trypan blue staining was used to analyze cytotoxicity caused by E1A and gemcitabine.

Immunoblotting.
Immunoblotting analyses were performed as described elsewhere (48 , 49) . Briefly, cell lysates were resolved by SDS-PAGE and then transferred to nitrocellulous membranes. The membranes were treated with an anti-E1A monoclonal antibody (NeoMarkers, Inc.) or anti-PARP polyclonal antibody (Oncogene Science), followed by incubation with a peroxidase-conjugated secondary antibody and detection with the enhanced chemiluminescence method (Amersham Pharmacia Biotech.).

Analysis of Apoptosis by Annexin-V-FLUOS Staining.
Cells (2 x 105) were plated in 6-cm tissue culture dishes. For analysis of AdE1A-mediated apoptosis, cells were treated with AdE1A(+) or AdE1A(-) (1 x 106 plaque-forming unit) for 12 h and next with 5 µg/ml gemcitabine for 24 h. Then, cells were trypsinized, washed twice with PBS, and processed for Annexin-V-FLUOS staining (Roche Molecular Biochemicals). For analysis of E1A-mediated proapoptotic activity, J5 parental cells and J5 E1A transfectants were treated with 5 µg/ml gemcitabine for 24 h. Then, cells were trypsinized, washed twice with PBS, and processed for Annexin-V-FLUOS staining (50) . The green fluorescence protein-stained cells were examined by fluorescence microscopy.

Analysis of NF-{kappa}B by the Electrophoretic Mobility Shift Assay.
Cells were treated with 5 µg/ml gemcitabine for 30 min. Then, cells were washed twice with PBS, scrapped into 1.5 ml of cold PBS, pelleted for 10 s, and then resuspended in 100 µl of cold buffer A [10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.2 mM phenylmethylsulfonyl fluoride]. Cells were allowed to swell on ice for 10 min and then vortexed for 10 s. Samples were centrifuged for 10 s, and the supernatant was discarded. The pellet was resuspended in 50 µl of cold buffer C [20 mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM phenylmethylsulfonyl fluoride] and incubated on ice for 20 min for high salt extraction. Cell debris was removed by centrifugation for 2 min at 4°C, and the supernatant fraction (nuclear extract) containing DNA-binding proteins was stored at -70°C. The nuclear extract (5 µg of protein) was incubated with 1 µg of poly (dI-dC) (Amersham Pharmacia Biotech.) on ice for 20 min, and then a 32P-labeled double-stranded oligonucleotide containing the {kappa}B site from the human immunodeficiency virus was added (51) . Binding of the radioactive oligonucleotide to nuclear DNA-binding proteins was carried out at room temperature for 20 min. The resulting oligonucleotide-protein complexes were resolved in 4% nondenaturing polyacrylamide gel.

Nuclear Run-on Assay.
The nuclear run-on assay was performed as described previously (52) . Briefly, cells were washed with PBS and lysed in ice-cold buffer [10 mM Tris (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% NP40]. The nuclei were then pelleted by centrifugation and suspended in buffer A [50 mM Tris (pH 8.3), 5 mM MgCl2, 0.1 mM EDTA, and 40% glycerol]. The nascent RNA chains were elongated by mixing the nuclear suspension with an equal volume of the reaction buffer [10 mM Tris (pH 8.0); 5 mM MgCl2; 0.3 M KCl; 5 mM DTT; 1 mM each of ATP, CTP, and GTP; and 0.1 mCi [{alpha}-32P]UTP; Amersham Pharmacia, Inc.]. After incubation at 30°C for 30 min, the 32P-labeled RNA was purified with TRIzol (Invitrogen, Inc.). Samples (5 µg) of plasmids, pCR3.1, pCR3.1-PARP, and pCR3.1-GAPDH were denatured and immobilized on Hybon N membranes. The membranes were prehybridized in a solution of 50% formamide, 5x saline-sodium phosphate-EDTA, 2x Denhardt’s reagent, 0.5% SDS, and 100 µg/ml salmon sperm DNA for 2 h at 42°C. Nascent RNA of equal radioactivity (1 x 106 cpm) from the test samples was hybridized with the immobilized DNA at 42°C for 24 h. Then, the membranes were washed for 60 min with 2x SSC and 0.1% SDS at 55°C, then for 30 min with 0.2x SSC and 0.1% SDS at 55°C, and finally exposed to X-ray film at -70°C.

Analysis of E1A + Gemcitabine-mediated Apoptosis in E1A-expressing Cells with Increased Expression of the p65 Subunit of NF-{kappa}B or PARP.
The full-length NF-{kappa}B p65 subunit and PARP cDNAs were synthesized by reverse transcription-PCR and cloned into an expression vector pCR3.1 plasmid (Invitrogen, Inc.). The primers for p65-NF-{kappa}B were forward 5'-ATGGACGAACTGTTCCCCCTC and reverse 5'-TTAGGAGCTGATCTGACTCAG; the primers for PARP were forward 5'-ATGGCGGAGTCTTCGGATAAG and reverse 5'-TTACCACAGGGAGGTCTTAAA. The cotransfection assay of a test plasmid and another plasmid containing a fluorescence gene was described previously (53) . Briefly, 1 x 106 JE-1 cells were plated in 6-cm tissue culture dishes and then transfected with a plasmid mixture [6 µg of pCR3.1-NF-{kappa}B and 0.6 µg of pDsRed or 6 µg of pCR3.1 vector and 0.6 µg of pDsRed as a control, i.e., the test plasmid: pDsRed plasmid = 10:1]. The pDsRed plasmid expresses a red fluorescent protein. After transfection (24 h), the cells were added a medium containing 5 µg/ml gemcitabine for 24 h and then subjected to Annexin-V-FLUOS staining (apoptosis assay as above described; a green fluorescent protein was tagged to Annexin V). The majority of red cells expressed NF-{kappa}B in case they were transfected with pCR3.1-NF-{kappa}B + pDsRed, because the ratio of pCR.1-NF-{kappa}B:pDsRed was 10:1. The green cells represented cells undergoing apoptosis. Both green and red cells were subjected to flow cytometry. The "percentage of apoptosis" was defined as the number of cells simultaneously with green and red colors divided by number of cells with red color only. The same assay was applied to PARP.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AdE1A Sensitizes HCC Cells to Gemcitabine.
To enhance the cytotoxicity of gemcitabine in HCC cells, we tested the synergistic effect of E1A and gemcitabine. J5, J7, Huh7, and HepG2 cells were treated with AdE1A(+) or AdE1A(-) and then with gemcitabine. Expression of E1A was detected by immunoblotting using an anti-E1A monoclonal antibody. The E1A gene encodes two proteins of 243 and 289 amino acids, resulting in doublets observed in Fig. 1Citation (Western blot analysis for E1A). Cells were treated with E1A-positive or -negative virus and then with different concentrations of gemcitabine. HCC cells, J5, J7, Huh7, and HepG2 pretreated with AdE1A(+) were more sensitive to gemcitabine (Fig. 1Citation ; 0.1–1 µg/ml gemcitabine). Cell number for each dot in Fig. 1Citation was normalized against the corresponding nongemcitabine-treated control. At 1 µg/ml gemcitabine, E1A resulted in an average 70% reduction in cell growth in comparison with non-E1A-expressing controls in which gemcitabine achieved only 35% reduction, suggesting a role of E1A as a chemosensitizer for gemcitabine in these cells.



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  		Fig. 1. Cytotoxic assay showing chemosensitizing effect of E1A on HCC cell lines. Top panels, Western blot analysis showing expression of E1A after adenoviral infection. Cells were subjected to Western blot 24 h after adenoviral infection. Expression of E1A was detected with anti-E1A monoclonal antibody. Ad.E1A(+) is an adenovirus with E1B and E3 deletions; AdE1A(-) is an adenovirus with E1A and E3 deletions. Bottom panels, enhanced cytotoxicity of gemcitabine to HCC cells by AdE1A(+) infection. Cells were infected with AdE1A(-) or AdE1A(+) and then treated with gemcitabine. Viable cells were counted by exclusion of trypan blue stained cells. Cell growth in response to gemcitabine during 72-h culture was calculated by the percentage of viable cells relative to the same cells without gemcitabine treatment. The values, expressed as percentages, have been normalized against the corresponding nongemcitabine-treated control cells. The experiment was done twice with similar results.

 
Characterization of E1A Transfectants of HCC Cell Lines.
Although the major genes that are responsible for propagation of adenovirus have been deleted in AdE1A(+) and AdE1A(-), the two viral genomes still contain other viral genes that may still have growth-suppressing effects resulting in tumor suppression not related to E1A. To simplify the effect of E1A, we established stable cell lines that expressed E1A. The E1A gene was cloned into the pSVneo vector and transfected into J5 and Huh7 cells. The stable transfectants were selected by G418. In J5 cells, 62 clones were picked, among which 12 clones had E1A expression. Three of these clones (JE-1, JE-2, and JE-3) were chosen for immunoblotting (Fig. 2A)Citation and other functional studies. Cellular sensitivity to gemcitabine was determined by trypan blue staining (Fig. 2B)Citation . JE series were more sensitive to gemcitabine than parental J5 and J5-C cells (J5-C is J5 cells transfected with pSVneo control vector). Using E1A transfectants to test the enhanced cytotoxic effect of gemcitabine by E1A, we obtained the same result as those from adenoviral infection. Therefore, we conclude that E1A enhances the cytotoxicity of gemcitabine in HCC cells. In addition, we also intended to establish stable E1A transfectants from Huh7 cells. More than 100 clones were picked out; 29 clones expressed E1A, but none of them grew well in culture. All of these E1A-positive Huh7 clones died after several passages, providing additional evidence of E1A’s role in tumor suppression of HCC cells.



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  		Fig. 2. Cytotoxic assay showing susceptibility of the E1A transfectants of J5 cells to gemcitabine. A, Western blot analysis showing expression of E1A in E1A transfectants of J5 cells. J5-C represents J5 cells stably transfected with the pSVneo vector. B, susceptibility of E1A transfectants to gemcitabine. Cells (J5, J5-C, and JE series) were treated with gemcitabine (0.1–1 µg/ml) for 72 h and then subjected to trypan blue staining as described in "Materials and Methods." Cell number was normalized as described in Fig. 1Citation . The experiment was performed twice with similar results.

 
Induction of Apoptosis by E1A in J5 Cells Treated with Gemcitabine.
In the above sections, we have demonstrated that E1A is a chemosensitizer for gemcitabine. To study the underlying mechanism, we examined whether E1A could induce J5 cells to apoptosis in the presence of gemcitabine. We adopted the Annexin-V-FLUOS staining from Roche Molecular Biochemicals to carry out this experiment. In the early stage of apoptosis, PS translocates from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the outer surface of the cell (50) . Annexin V is a Ca2+-dependent phospholipid-binding protein with high affinity for PS. The protein can be used as a sensitive probe for PS exposed on the outer leaflet of the cell membrane and is hence suited to detect apoptotic cells (50) . J5 cells were treated with AdE1A(+) or AdE1A(-) for 12 h and next with gemcitabine for 24 h. Then, cells were stained with green fluorescent protein-labeled Annexin V and analyzed by fluorescence microscopy (Fig. 3)Citation . There were no apoptotic cells observed in J5 cells treated with gemcitabine (Fig. 3Citation , panel 1). AdE1A(-)-infected J5 cells did not go to apoptosis in the presence of gemcitabine (data not shown). J5 cells treated with AdE1A(+) alone did not go to apoptosis either (Fig. 3Citation , panel 2), but AdE1A(+)-infected cells went to apoptosis while exposed to gemcitabine (green fluorescent cells in panel 3). The E1A transfectant JE-1 was also subjected to the same test. JE-1 cells were treated with gemcitabine for 12 h and then subjected to the Annexin-V-FLUOS staining (Fig. 3Citation , panel 5; panel 4 as a control). JE-1 cells were induced to apoptosis while treated with gemcitabine. This study suggests that the proapoptotic activity of E1A contributes to the enhanced cytotoxicity observed in Figs. 1Citation and 2Citation .



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  		Fig. 3. E1A induces J5 cells to apoptosis in the presence of gemcitabine. J5 cells were treated with one of the adenoviruses used in this study or gemcitabine (GMT) or an adenovirus + gemcitabine. Apoptosis was analyzed by Annexin-V-FLUOS staining. In panel 1, apoptosis was not induced in J5 cells during treatment with 5 µg/ml gemcitabine for 24 h. Apoptosis was not induced in J5 cells during treatment with AdE1A(-) or AdE1A(-) + gemcitabine as well (data not shown). Panel 2, apoptosis was not induced in J5 cells during treatment with AdE1A(+) alone. Panel 3, apoptosis was induced in J5 cells with during treatment AdE1A(+) + gemcitabine. Panel 4, JE-1 cells as a control for Panel 5, which shows apoptosis in JE-1 cells treated with gemcitabine.

 
Suppression of NF-{kappa}B Activity by E1A in HCC Cells Treated with Gemcitabine.
We have reported previously that E1A suppresses NF-{kappa}B activation in SKOV3.ip1 cells treated with {gamma}-irradiation or TNF-{alpha} (42 , 43) . In the above study, we have shown that E1A triggers apoptosis in HCC cells treated with gemcitabine. These findings raise a possibility that gemcitabine may induce NF-{kappa}B activation, which is suppressed by E1A. To test this hypothesis, we treated J5 cells and the same cells expressing E1A with gemcitabine and then analyzed NF-{kappa}B activity by electrophoretic mobility shift assay. As shown in Fig. 4ACitation , gemcitabine activated NF-{kappa}B in J5 cells (lanes 2 and 6, the p65 and p50 heterodimer of NF-{kappa}B) but not in the cells expressing E1A (lanes 4 and 8). A supershift from the complex of the radioactive oligonucleotide and NF-{kappa}B heterodimer was observed in the nuclear extract from gemcitabine-treated J5 cells, to which was added an anti-p65 subunit antibody, giving further evidence that the shifted bands in lanes 2 and 6 were from NF-{kappa}B binding. This result is consistent with our previous report that E1A suppressed NF-{kappa}B activation in SKOV3.ip1 cells in response to apoptotic stimuli, such as {gamma}-irradiation and TNF-{alpha} (42 , 43) , suggesting a role of NF-{kappa}B suppression in promotion of gemcitabine-induced cell death by E1A.



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  		Fig. 4. In A, suppression of NF-{kappa}B activation by E1A. J5 cells with or without E1A expression were treated with 5 µg/ml gemcitabine and then subjected to electrophoretic mobility shift assay for NF-{kappa}B activity. Lane 1, J5-C cells; Lane 2, J5-C cells treated with gemcitabine; Lane 3, JE-1 cells; Lane 4, JE-1 cells treated with gemcitabine; Lane 5, J5 cells infected with AdE1A(-); Lane 6, J5 cells infected with AdE1A(-) and then treated with gemcitabine; Lane 7, J5 cells infected with AdE1A(+); Lane 8, J5 cells infected with AdE1A(+) and then treated with gemcitabine; Lane 9, J5 cells treated with gemcitabine and then subjected to NF-{kappa}B assay, to which was added an anti-p65 subunit of NF-{kappa}B antibody (Santa Cruz Biotechnology). In B, increased expression of p65 NF-{kappa}B in E1A-expressing J5 cells overcomes E1A+gemcitabine-mediated apoptosis. Cells were transfected with p65 NF-{kappa}B expression vector and pDsRed at a ratio of 10:1, treated with gemcitabine, and then subjected to apoptosis assay by Annexin-V-FLUOS staining. Column 1, JE-1 cells transfected with control vector and then treated with gemcitabine; column 2, JE-1 cells transfected with p65 NF-{kappa}B expression vector and then treated with gemcitabine; column 3, J5 cells infected with AdE1A(+), transfected with control vector, and then treated with gemcitabine; column 4, J5 cells infected with AdE1A(+), transfected with p65 NF-{kappa}B expression vector, and then treated with gemcitabine. The value in each column was the mean of triplicate samples.

 
To study whether exogenous expression of NF-{kappa}B by gene transfection would circumvent the effect of E1A, the JE-1 cells were transfected with a plasmid mixture containing plasmids pCR3.1-NF-{kappa}B (p65) and pDsRed at a ratio of 10:1 and then treated with gemcitabine. Apoptotic cells were analyzed by Annexin-V-FLUOS staining (apoptotic cells shown with green fluorescence). Because the ratio of pCR3.1-NF-{kappa}B:pDsRed plasmid used for cotransfection experiments is 10:1, the majority of cells expressing red fluorescent protein would express exogenous p65 NK-{kappa}B. After plasmid transfection and gemcitabine treatment, cells were subjected to flow cytometry. The number of cells with green and red fluorescence over that of cells with only red fluorescence represented the percentage of apoptotic cells. As shown in Fig. 4BCitation , both JE-1 (a stable transfectant of E1A) cells and AdE1A(+) virus-infected J5 cells with pCR3.1-p65 NF-{kappa}B transfection showed a 30% decrease in cells undergoing apoptosis than those with pCR3.1 vector transfection, suggesting a role of NF-{kappa}B in protection of E1A+gemcitabine-mediated apoptosis. The transfection efficiency was ~10–15%, which was obtained from the data of flow cytometry. The values accounting for each column in Fig. 4BCitation were adjusted by transfection efficiency. The differences between columns 1 and 2 and 3 and 4 were significant through Student’s t test (P < 0.05).

Suppression of PARP Expression by E1A in HCC Cells Treated with Gemcitabine.
In the course of apoptosis, the caspase cascade is activated, leading to cleavage of essential proteins required for cell survival (54) . Caspase-3 and caspase-7 cleave PARP into an Mr 89,000 COOH-terminal fragment, with a reduced catalytic activity, and a Mr 24,000 NH2-terminal peptide, which retains the DNA binding domains during apoptosis (55 , 56) . To study whether E1A + gemcitabine would induce PARP cleavage, cells (with or without E1A expression) were treated with gemcitabine (5 µg/ml) for 24 h and then subjected to Western blotting. Gemcitabine induced the expression of PARP; however, E1A + gemcitabine did not induce cleavage of PARP. Expression of PARP was suppressed by E1A. The Mr 24,000 peptide derived from PARP cleavage by caspase-3 or caspase-7 was not prominent in cells undergoing apoptosis (Fig. 5A)Citation . To further examine the underlying mechanism, we did nuclear run-on assay to determine the level at which E1A affected PARP expression. As shown in Fig. 5BCitation , gemcitabine induced transcription of PARP, and cells expressing E1A showed decreased mRNA transcription of PARP (the former: latter ~10:1). This study demonstrates that E1A suppresses PARP expression at the transcriptional level rather than induces cleavage of the protein in apoptotic cells.



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  		Fig. 5. A, decreased expression of PARP in E1A-expressing cells. Cells with or without E1A expression were treated with gemcitabine and then subjected to immunoblotting by anti-PARP polycolonal antibody. The same transblot was reprobed with anti-{alpha}-actin antibody. In B, E1A represses PARP expression at the transcriptional level. Cells with or without E1A expression were treated with gemcitabine and then subjected to the nuclear run-on assay as described in "Materials and Methods." In C, reexpression of PARP in E1A-expressing J5 cells overcomes E1A+gemcitabine-induced apoptosis. Cells were transfected with a PARP expression vector (or the control vector pCR3.1) and pDsRed at a ratio of 10:1, treated with gemcitabine, and then subjected to apoptosis assay by Annexin-V-FLUOS staining. Column 1, JE-1 cells transfected with control vector and then treated with gemcitabine; column 2, JE-1 cells transfected with PARP expression vector and then treated with gemcitabine; column 3, J5 cells infected with AdE1A(+), transfected with control vector, and then treated with gemcitabine; column 4, J5 cells infected with AdE1A(+), transfected with PARP expression vector, and then treated with gemcitabine. The value of each column was the mean of triplicate samples.

 
To determine whether PARP is able to protect cells from E1A+gemcitabine-mediated apoptosis, JE-1 cells were transfected with pCR3.1-PARP and pDsRed and then subjected to the apoptosis assay (Annexin-V-FLUOS staining) used for NF-{kappa}B (Fig. 4B)Citation . As shown in Fig. 5CCitation , JE-1 cells expressing exogenous PARP showed a 65% decrease in apoptosis. Similar results were obtained from J5 cells infected with AdE1A(+). The data processing was the same as in p65 NF-{kappa}B transfection assay. The differences between columns 1 and 2 and 3 and 4 in Fig. 5CCitation were significant through Student’s t test (P < 0.05). These results suggest that increased PARP is required for prevention of cells from E1A+gemcitabine-mediated apoptosis.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
HCC has put medical oncologists in a bitter predicament for a long time. The response rate of HCC to local or systemic chemotherapy has been especially disappointing (6, 7, 8, 9, 10) . The cytidine analogue gemcitabine has shown strong antitumor effects on a variety of human cancers (11, 12, 13, 14) . A few clinical trials have also shown that the agent has antitumor activity for human HCC (21, 22, 23, 24) ; however, the median survival times in the published reports are still very short. It appears that gemcitabine monotherapy cannot be recommended for use in HCC. Because HCC is highly resistant to currently available anticancer drugs, combination therapy with other anticancer drugs, although worth trying, might eventually give dismal outcomes.

The progress of gene therapy was rapid over the past decade and has shed light on cancer treatment. Of the genes under study, AdE1A might be the candidate gene best studied for cancer therapy. E1A is a tumor suppressor, and E1A gene transfer has resulted in increased cell sensitivity to paclitaxel (Taxol) in human breast cancer (38) and to cisplatin and etopside in sarcoma cells (41) . In this report, we show that E1A acts as a tumor suppressor for HCC cells and sensitizes these cells to gemcitabine. Although J5 cells expressing E1A were still able to proliferate in cell culture, they grew slowly in culture and lost the ability to induce tumor in animals (data not shown). The established E1A transfectants of J5 cells were subjected to aging. As observed in E1A transfectants of Huh7 cells, J5 clones stably expressing E1A, although having longer passage time than E1A-transfected Huh7 cells, died eventually. Thus far, we still have difficulty establishing permanent E1A stable transfectants from J5 and Huh7 cells. E1A may produce these effects by derepressing epithelial promoters through inhibition of nonepithelial transcriptional factors (57) . Anoikis, an apoptotic change induced by disruption of the interactions between normal epithelial cells and extracellular matrix, may also be a possible mechanism for E1A-mediated tumor suppression (58) . In an animal study, E1A mediates a bystander effect on tumor suppression by inhibiting angiogenesis (59) . The mechanism of E1A-mediated tumor suppression is multifactorial. New mechanisms are still under study now.

As stated above, gemcitabine apparently has antitumor activity for HCC, although its efficacy remains to be improved. In the current study, we made an attempt to increase its efficacy by E1A gene transfer. E1A by itself possessed antitumor activity for HCC cells (Fig. 1)Citation . In the presence of E1A, gemcitabine had greater cytotoxicity in HCC cells (Fig. 1)Citation . Therefore, transfer of E1A into HCC cells in combination with gemcitabine is a potential strategy to treat the malignancy. We also studied the underlying mechanism and found that E1A had proapoptotic activity, resulting in a tendency of HCC cells to undergo apoptosis while exposed to gemcitabine. Taken together, introduction of E1A into HCC cells indeed increases the effectiveness of gemcitabine. E1A may be potentially used as a therapeutic gene in combination with gemcitabine to treat HCC.

E1A has been put on clinical trials for breast and head and neck cancers. The published reports have given promising results (34 , 35) . The Phase I trial of E1A gene therapy was conducted in the University of Texas M. D. Anderson Cancer Center by intracavitary injection of the E1A gene in patients with both HER-2/neu-overexpressing and low HER-2/neu-expressing breast and ovarian cancers. This E1A gene expression was accompanied by HER-2/neu down-regulation, increased apoptosis, and reduced proliferation. The most common treatment-related toxicities were fever, nausea, vomiting, and/or discomfort at the injection sites (34 , 35) . In another multicenter Phase I trial, 16 patients of metastatic breast or head and neck cancer were evaluated after intratumor injection of the E1A gene; 2 had minor responses, 8 had stable disease, and 6 had progressive disease (35) . As yet, there is no major toxicity noted in patients who are treated with E1A. It appears that cytotoxicity of E1A gene therapy to normal cells is lower than conventional chemotherapy. Clinical investigators who work on E1A gene therapy continue to monitor its toxicity to the human beings.

The initial molecular mechanism of E1A-mediated tumor suppression was down-regulation of Her-2/neu proto-oncogene (26, 27, 28, 29) . However, the mechanism for E1A-mediated tumor suppression in non-Her-2/neu-expressing cells remains to be determined. Suppressing NF-{kappa}B activation has been shown to play a role (42 , 43) . NF-{kappa}B is inactive in association with unphosphorylated I{kappa}B in the cytoplasm. In response to harmful stimuli, such as TNF-{alpha}, {gamma}-irradiation, and cytotoxic agents, I{kappa}B is phosphorylated and then degraded rapidly by the ubiquitin degradation system, leading to activation of NF-{kappa}B, which translocates into the nucleus and functions as a transcriptional factor for the expression of survival genes. The activated NF-{kappa}B is mainly composed of two subunits, i.e., p50 and p65, and has been shown to have antiapoptotic activity, particularly the p65 subunit that is the cellular homologue of the viral oncogene Rel A (60 , 61) . Suppression of NF-{kappa}B activation is one of the mechanisms by which E1A promotes apoptosis in cells exposed to apoptotic stimuli (42 , 43) . The mechanism is through stabilization of I{kappa}B in the unphosphorylated form, which binds to NF-{kappa}B. In this study, we demonstrate that gemcitabine induces NF-{kappa}B activation, which is suppressed by E1A. In addition, we also show that increased expression of the p65 subunit of NF-{kappa}B counteracts the E1A+ gemcitabine-mediated apoptosis.

PARP is a zinc-finger DNA-binding protein, which detects DNA breaks generated during base excision repair or by genotoxic agents. In response to genotoxic agents, PARP catalyzes the synthesis of poly (ADP-ribose) from ß-NAD+; this polymer is covalently attached to several nuclear proteins and PARP itself. As a result, PARP converts DNA breaks into intracellular signals, which activate DNA repair or cell death programs (62 , 63) . Several studies have also shown that PARP is involved in the fate of cells, e.g., necrosis, inflammation, or apoptosis in response to cytotoxic stimuli (64, 65, 66) . PARP is activated at an intermediate stage of apoptosis and is then cleaved and inactivated at a late stage by apoptotic proteases, namely caspase-3 and -7. This cleavage may prevent necrosis during the repair process after ischemia of the heart and brain, avoiding acute inflammation (64, 65, 66) . We have shown in Fig. 4Citation that E1A sensitizes J5 cells to apoptosis in the presence of gemcitabine. Thus, we initially thought that E1A would induce PARP cleavage in cells treated with gemcitabine. However, the experimental results were beyond our expectation. Rather, E1A inhibited PARP expression at the transcriptional level (Fig. 5B)Citation . Re-expression of PARP protects E1A-expressing cells from E1A + gemcitabine-mediated apoptosis (Fig. 5C)Citation . Gemcitabine-triphosphate is a nucleotide analogue, which can be incorporated into DNA, resulting in chain termination during DNA synthesis. It appears that gemcitabine-treated J5 HCC cells increase PARP expression by instinct to overcome (at least delay) gemcitabine-induced DNA damage. Our results give a reasonable explanation why E1A sensitizes HCC cells to gemcitabine. The recent studies have suggested that neuronal and heart muscle cells may undergo necrosis in response to prolonged hypoxia; however, inhibition of PARP shifts necrosis to apoptosis (67 , 68) . However, studies focused on cancer cells have given different conclusions: (a) inactivation of PARP sensitized cells to the anticancer drug CHS 828 (69) ; and (b) caspase-resistant PARP prevented etopside-mediated DNA fragmentation through down-regulation of endonuclease DNAS1L3 (70) . The two studies in cancer research support our model that E1A represses the expression of PARP and thereby sensitizes cells to gemcitabine. We speculate that there are divergent PARP functions between cancer and normal cells while they are rendered to apoptosis.

Establishing an effective systemic therapy for HCC has been a frustrating challenge. Doxorubicin is the wildly used single agent for HCC therapy (71 , 72) . However, among the largest studies using doxorubicin for patients with advanced disease, the median survival time has been <4 months. Newly developed agents, including paclitaxel (73) , irinotecan (74) , raltitrexed (75) , and nolatrexed (76) , have also given disappointing results. The trials of gemcitabine on HCC treatment, although showing improved median survival times in some studies, have not made a breakthrough advancement in the disease’s outcome. Here, we offer an in vitro study in enhancing cytotoxicity of gemcitabine and provide underlying mechanisms, i.e., E1A suppresses the activity of NF-{kappa}B and expression of PARP, which at least in part results in accelerated apoptosis in HCC cells treated with gemcitabine and E1A.


   ACKNOWLEDGMENTS
 
We thank Dr. Yun-Fan Liaw, the founder of the Liver Research Unit in Chang Gung Memorial Hospital, for selfless giving of his love to the research group.


   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 Supported by National Science Council, Taiwan (NSC90-2314-B-182A032 to W-P. L.; NSC91-2315-B-182A003 to D-I. T.), Chang Gung Memorial Hospital (CMRP 1061 to W-P. L.), and Taipei Veterans General Hospital (A-92027 to W-P. L.). Back

2 To whom requests for reprints should be addressed, at Department of Internal Medicine, Taipei Veterans General Hospital, 201 Shi-Pai Rd Sec 2, Taipei, Taiwan. Fax: 011-886-2-2874-9425; E-mail: wleemc{at}yahoo.com Back

3 The abbreviations used are: HCC, hepatocellular carcinoma; AdE1A, adenovirus type 5 E1A; dFdC, 2',2'-diflurodeoxycytidine; PS, phosphatidylserine; PARP, poly(ADP-ribose) polymerase; NF-{kappa}B, nuclear factor-{kappa}B; TNF, tumor necrosis factor. Back

Received 9/11/02. Revised 6/24/03. Accepted 7/23/03.


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 DISCUSSION
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