Adenovirus E1A Expression Enhances the Sensitivity of an Ovarian Cancer Cell Line to Multiple Cytotoxic Agents through an Apoptotic Mechanism1

The introduction of adenovirus 5 EJA into the SKOV3ip1 ovarian cancer cell line was shown previously to suppress HER2/neu expression and reduce the malignant potential of these cells (Vu et a!., Cancer Res., 53: 891-898, 1993). In this report, we show that reduction of p185 in cells stably expressing E1A protein was coincident with increased sensitivity to cytotoxic agents. The LD50 of cisplatin was reduced 6-fold, and the LD50 of paclitaxel and doxorubicin was reduced 10-fold in E1A-expressing cells compared with control cells. The growth of SKOV3ip1 and control cells was unchanged in the presence of 150 ng/ml of tumor necrosis factor-a, whereas the growth of E1A-expressing cells was reduced by 30 to 40%. When we used a physiologically obtainable concentration of paclitaxel (0.5 ELM), DNA bad. dering consistent with apoptotic cell death was seen after a 24-h exposure in the E1A-expressing cells, whereas laddering and DNA fragmentation were only detected in DNA from control cells after longer exposure ( 48 h) at a 20-fold higher concentration of paclitaxel. The SKOV3ip1 cells do not express p53 protein; hence, the induction of apoptosis by paclitaxel is through a p53-independent pathway. Despite their diverse mechanisms of action, the cytotoxic effects of cisplatin, doxorubicin, paclitaxel, and tumor necrosis factor-a were enhanced by the expression of E1A proteins in the SKOV3ip1 ovarian cancer cells. This suggests that these agents share a common final pathway of cell killing, which may represent a potential therapeutic target in resistant ovarian cancers. Received 10/23/96; revised 7/1 1/97; accepted 7/11/97. 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. I This work was supported in part by a Texas Division Oncology Fellowship from the American Cancer Society (to K. R. B.) and Grant CA51053 from the National Cancer Institute, NIH (to J. E. P.). 2 To whom requests for reprints should be addressed, at Department of Cell Biology, Box 173, The University ofTexas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-8212; Fax: (713) 792-8747. INTRODUCTION The adenovirus 5 early region la (EJA) gene products are multifunctional transcriptional regulators capable of interacting with and perturbing the function of key regulators of cell growth. The primary function of the EJA gene products is to activate other adenoviral genes during a permissive viral infection by modifying the transcriptional apparatus of the host cell. EJA functions in concert with EJB to regulate the expression of viral genes in transformed cells (1), although expression of EJA alone predisposes such cells to apoptosis. EJA-induced apoptosis has been reported to be dependent on the presence of wildtype p53 protein (2) and can be inhibited by E1B proteins (2, 3) or bcl-2 (4). EJA expression can sensitize certain cells to apoptosis induced by various cytotoxic agents. Induction of EJA expression has been shown to increase the susceptibility of myeloid leukemia cells to lysis by TNF3-a (5). Transfection of a fibroblast cell line with EJA sensitized these cells to apoptosis induced by ionizing radiation and by chemotherapeutic agents that have widely divergent mechanisms of action. This cell death was regarded as p.53 dependent, because p.53 null cells did not show enhanced sensitivity after EJA transfection (6). Systemic chemotherapy is the standard treatment for metastatic ovarian cancer. Despite improved median survival for patients with advanced stage disease treated with cisplatin and, more recently, paclita.xel, the development of resistance remains a major obstacle to improving overall survival. The initial response rates to these agents can be as high as 80-90%, but the majority of patients relapse (7). Response rates to second-line agents are 25-30%, with few durable responses (8). It appears that the development of resistance to the first-line agents cisplatin and paclitaxel is indicative of a general unresponsiveness to further cytotoxic therapy. Much attention has been focused on developing new agents with unique mechanisms of action, but chemotherapy resistance in ovarian cancer may not be occurring at the level of the drug-target interaction. Instead, a downstream process may be involved. A defect or alteration in the apoptotic pathway could lead to resistance to multiple cytotoxic agents. Indeed, defects in the apoptotic pathway have been shown to produce treatment-resistant tumors in vivo (9). Restoration or enhancement of apoptosis in drug-refractory tumor populations could potentially prevent or reverse drug resistance. In this study, we report that the human ovarian cancer cell line, SKOV3ipl, was found to be relatively resistant to various cytotoxic agents (cisplatin, paclitaxel, doxorubicin, and TNF-a). 3 The abbreviations used are: TNF, tumor necrosis factor: FBS, fetal bovine serum; MU, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazohum bromide; FACS, fluorescence-activated cell sorting; DPA, diphenylamine. Research. on April 13, 2017. © 1997 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 2018 Adenovirus EJA and Chemosensitivity The introduction of the EJA gene, which we showed previously could suppress expression of HER2/neu and reduce malignant growth potential of these cells (10), resulted in increased sensitivity to all of these agents, which act through different modes of action. For paclitaxel treatment, the increased sensitivity was associated with enhanced induction of DNA fragmentation, one indicator of apoptosis. The SKOV3 and variant cell lines were all p.53 null, and thus the EJA-enhanced apoptosis observed must occur through a p53-independent mechanism. MATERIALS AND METHODS Cell Lines. The human ovarian cancer cell line SKOV3 was obtained from the American Type Culture Collection. The SKOV3ipl cell line was derived from the ascites of a nude mouse injected with the original cell line (10). As described previously, this cell line was transfected with either the EJAexpressing plasmid (pE1A) or pElA-d1343, which contains a two-base frameshift deletion in the EJA coding region. The transfection mixtures also contained pSV2neo plasmid to confer resistance to G4l8 (10). The cell line and derivatives were maintained in monolayer culture in Eagle’s MEM supplemented with 5% FBS, 1 mM sodium pyruvate, 0. 1 mrvi nonessential amino acids, I mM L-glutamine, 50 units/mI penicillin G, 50 i.g/ml streptomycin sulfate and MEM vitamin supplement (medium and supplements from Life Technologies, Inc., Grand Island, NY). The transfected cells were maintained in medium containing 800 p.g/ml G4l8 (Life Technologies, Inc.). Cells were harvested by incubation with 0.25% trypsin-0.02% EDTA and suspended in MEM supplemented with 5% FBS. Measurement of in Vitro Growth Rates and Serum Dependence. The growth rates of the SKOV3 variants and transfectants in medium with different concentrations of FBS were measured to assess how the presence of EJA affected cell proliferation. The SKOV3, SKOV3ip1, one EIA-transfected clone, ElAl7, and the control transfected line, EFS, were plated in 96well plates, 5 x l0 cells/well in MEM with 5% FBS. After 1-5 days incubation, the relative cell number was determined using the MU assay ( 1 1). To assess the serum dependence of the cell lines, the cells were initially plated in 96-well plates, as above, in MEM with 5% FBS. After 24 h incubation to allow the cells to attach completely, the medium was aspirated, the wells were washed twice with serum-free MEM, and medium was supplemented with 0 to 5% FBS added. After a 6-day incubation period, the relative cell numbers were determined using the MU assay. Chemosensitivity Assays. Cells were plated in 96-well plates, 5 X l0 cells/well. After 24 h incubation, the medium was aspirated and replaced with MEM with 5% FBS containing a range of concentrations of r-hu-TNF-a (Genzyme, Cambridge, MA), cisplatin (Bristol-Myers Oncology Division, Syracuse, NY), doxorubicin (Ben Venue Laboratories Inc., Bedford, OH), or paclitaxel (Sigma Chemical Co., St. Louis, MO), and incubated for an additional 72 h. Relative numbers of viable cells remaining were determined using the MTT assay. In assays for sensitivity to TNF-a, the mouse L929 fibroblast cell line served as a positive control, because these cells are exquisitely sensitive to this cytokine (12). Western Blot Analyses for E1A and p53 Proteins. Protein lysates were prepared from cultures of cells in the logarithmic phase of growth. Aliquots of 10 pg of protein were separated electrophoretically on 10% SDS-polyacrylamide gels and transferred to nitrocellulose filters. For E1A expression, the filters were incubated for 1 h at room temperature with mouse monoclonal antibody M73 (Santa Cruz Biotechnology, Santa Cruz, CA). For p53 protein analysis, the filters were hybridized with the DO-I mouse monocbonal antibody (Santa Cruz Biotechnology), which recognizes the N1-I2-terminal epitope of wild-type and mutant human p53 protein. The filters were incubated with anti-mouse horseradish peroxidase-conjugated antibody and developed with ECL detection reagents (Amersham Corp., Arlington Heights, IL). To confirm equal loading and transfer of protein, the filters were stripped and re-probed with a rabbit anti-actin antibody (Sigma Chemical Co.) and anti-rabbit horseradish peroxidase-conjugated antibody (Amersham). Densitometric calculations of the intensity of bands corresponding to specific proteins were performed using a Molecular Dynamics Personal Densitometer. FACS Analysis. Cells grown to 70 to 80% confluence were harvested with trypsin and EDTA and suspended in PBS with 1% FBS at 1 X 106 cells per ml. Monocbonal anti-human p185 (Ab5, Oncogene Science, Uniondale, NY) was added at a final dilution of 1:250 and incubated at 4#{176}C for 30 mm. The cells were washed with PBS containing 2% FBS and 0.02% sodium azide, then incubated with FITC-conjugated goat F(ab’), anti-mouse IgG (Biosource, Camarillo, CA) at 1:20 dilution, at 4#{176}C for 30 mm. The cells were washed extensively and fixed in 1% paraformaldehyde in PBS. FACS analysis was performed on an EPICS Profile Cell Sorter (Coulter, Hileah, FL) with a 525-nm band pass filter to detect FITC and gated on forward versus side scatter to exclude debris, dead cells, and clumps. Analysis was based upon cursors set at 2% for isotype-matched negative controls. DNA Laddering. Cells were plated in 150-cm2 tissue culture flasks to 70% confluence, and paclitaxel was added to the medium at the calculated LD99 doses, 0.5 p.M for E1A17 cells and 10 iM for EFS cells. After 24 h incubation, the cells were harvested with trypsin-EDTA and counted. A portion of the cells was used for the DPA assay (see below). Aliquots of 106 cells were suspended in 0.3 ml of lysis buffer ( I % sarcosyl, 0.01 M EDTA, and 10 mg/ml RNase A in PBS) and incubated for 4 h at 37#{176}C. Proteinase K was added to a final concentration of 100 ig/ml and incubated at 50#{176}C for 12 h. High molecular weight DNA was pelleted by high speed centrifugation, and the supernatant was extracted twice with equal volumes of phenol: chloroform:isoamyl alcohol (25:24: 1). Following the addition of 1/10 final volume of 2 M sodium acetate and 2 volumes of 100% ethanol to the aqueous supernatant and freezing at 70#{176}C for 2 h, the precipitated DNA was pelleted, washed with 70% ethanol, and air dried. The DNA was then dissolved in 50 i.l of TE buffer (10 msi Tris-l mM EDTA) and separated electrophoretically in a 1% agarose/ethidium bromide gel at 20 V for 15 h. The gel was visualized under UV illumination and photographed. Diphenylamine Reaction. The DPA reaction was used to quantify DNA fragmentation. In this assay, fragmented DNA released from the nuclei of lysed cells is separated from intact chromatin by centrifugation. The intact and fragmented DNA is quantified by measuring deoxyribose using the DPA colorimetric assay (13). The E1A17 and EFS cells were treated with paclitaxel for up to 72 h, as described above, and harvested with trypsin-EDTA. Aliquots of 5 X 106 cells in 0.5 ml of PBS were Research. on April 13, 2017. © 1997 American Association for Cancer clincancerres.aacrjournals.org Downloaded from