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E1A Sensitizes HER2/neu-overexpressing Ewing’s Sarcoma Cells to Topoisomerase II-targeting Anticancer Drugs¹

Department of Cancer Biology, Pediatrics and Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effect of E1A on... Effect of HER2/neu on... Effect of E1A Gene... DISCUSSION REFERENCES

 
Overexpression of the HER2/neu oncogene is associated with tumorigenicity and drug resistance in many types of cancer. Three different human Ewing’s sarcoma cell lines (TC71, RD, and A4573) were found to express high levels of the HER2/neu protein. Transduction of TC71 cells with the E1A gene using an adenoviral vector (Ad-E1A) down-regulated HER2/neu overexpression in those cells and increased cytostasis. E1A-induced apoptosis was demonstrated by both flow cytometric analysis and Western blot analysis using a poly(ADP-ribose) polymerase antibody. After transduction of the E1A gene into these cells, the sensitivity of these cells to VP-16 (etoposide) was enhanced 18-fold and to Adriamycin 5-fold. However, no change was seen in cisplatin sensitivity. E1A also significantly increased topoisomerase II protein expression, indicating that the up-regulation of topoisomerase II may be one of the mechanisms by which E1A enhanced the sensitivity to topoisomerase II-targeting anticancer drugs, such as VP-16 and Adriamycin, but not cisplatin. In summary, these studies demonstrated that Ad-E1A can down-regulate HER2/neu overexpression and up-regulate topoisomerase II expression in human Ewing’s sarcoma cells, increasing their apoptosis rate and enhancing their sensitivity to VP-16 and Adriamycin.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effect of E1A on... Effect of HER2/neu on... Effect of E1A Gene... DISCUSSION REFERENCES

 
Ewing’s sarcoma is the second most common malignant bone tumor in children (1) . The majority of patients have microscopic metastases at diagnosis; the lung is the most common metastatic site (2) . These patients initially respond to systemic chemotherapy plus surgery and/or radiation. However, at 2 years, 40–60% will, depending on the prognostic indicators, have a recurrence. Adverse prognostic indicators include large tumor volume or tumor length, tumor location, elevated serum lactic dehydrogenase, and metastatic spread to bone, lung, or bone marrow. A number of different chemotherapy regimens, including those comprising total-body irradiation and autologous bone marrow transplant, have been unsuccessful in increasing the survival rates at 2 and 3 years (3, 4, 5, 6, 7) . Furthermore, relapsed patients are particularly resistant to second-line chemotherapy, leaving few treatment options.

The HER2/neu oncogene encodes a M_r 185,000 human epidermal growth factor receptor-2 transmembrane protein. Overexpression of HER2/neu has been found in 30% of human breast and ovarian cancers (8 , 9) . Enhanced expression of HER2/neu increased the tumorigenicity and metastatic potential of human cancer cells (10 , 11) . Clinical studies indicate that overexpression of HER2/neu correlates with poor prognosis, shorter patient survival in osteosarcoma (12 , 13) . Because of the close association reported between overexpression of HER2/neu and resistance to chemotherapeutic agents (14 , 15) and because of the poor disease-free survival rates associated with multi-agent chemotherapy in Ewing’s sarcoma, we investigated HER2/neu expression in several different Ewing’s sarcoma cell lines. HER2/neu expression was high in all three of the Ewing’s sarcoma cell lines examined.

Down-regulation of HER2/neu may be an important approach for cancer therapy (16 , 17) . The E1A³ gene has been shown to be a tumor suppressor for cells that overexpress HER2/neu (18 , 19) . Transfer of the E1A gene results in transcriptional repression of HER2/neu via an effect on the HER2/neu promoter. In vivo, E1A gene transfer suppressed the dissemination of ovarian cancer cells that overexpressed HER2/neu and enhanced the in vivo sensitivity of the cells to paclitaxel (20 , 21) . In this study, we found that E1A down-regulated HER2/neu expression, inhibited tumor cell growth, and increased cellular sensitivity to topoisomerase II-directed anticancer drugs. These results suggest that E1A gene therapy aimed at down-regulation of HER2/neu may provide a new approach for the therapy of Ewing’s sarcoma.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effect of E1A on... Effect of HER2/neu on... Effect of E1A Gene... DISCUSSION REFERENCES

 
Cell Lines.
TC71 human Ewing’s sarcoma cells, kindly provided by Dr. P. Pepe (University of Southern California, Los Angeles, CA), were cultured in Eagle’s modified essential medium with 10% fetal bovine serum. SKRB-3 human breast cancer cells and RD human Ewing’s sarcoma cells were obtained from American Type Culture Collection (Manassas, VA). A4573 human Ewing’s sarcoma cells were a generous gift from Dr. Soldatenkov (Georgetown University Medical Center, Washington, DC) and were cultured in RPMI 1640 with 15% fetal bovine serum. Normal human osteoblast cells were purchased from Clonetics, Inc. (San Diego, CA) and were maintained in the special medium provided by Clonetics. All cells were free of Mycoplasma as screened by Gen-Probe (Gen-Probe, San Diego, CA).

Recombinant Adenovirus.
Ad-E1A is an adenovirus type 5 that contains E1A but lacks E1B and E3 (18) . The control adenovirus, Ad-ß-gal, is an adenovirus type 5-based vector that lacks E1A, E1B, and E3 but contains ß-gal. Both of these recombinant replication-deficient adenoviral vectors were propagated in 293 cells as described previously (22) . The viruses were purified twice using cesium chloride-gradient ultracentrifugation and then dialyzed and titrated by the standard method. Cells were infected with adenovirus at 10 plaque-forming units/cell for 48 h and then treated as indicated for various experiments.

Cytostasis Assay.
Cells were seeded into 96-well cell culture plates (5000/well) and allowed to adhere overnight before being infected with Ad-E1A or Ad-ß-gal. Various concentrations of VP-16, Adriamycin, or cisplatin were then added in triplicate. The antiproliferative activity was determined 48 h later by the MTT assay as described previously (23) .

Western Blot.
Cells were set in 100-mm dishes (2 x 10⁶ per dish) 1 day before treatment and then were infected with Ad-E1A or Ad-ß-gal for 48 h. Cells were washed with cold PBS buffer and lysed in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS in PBS) containing the fresh protease inhibitor aprotinin (2 µg/ml), leupeptin (2 µg/ml), pepstatin A (1 µg/ml), and phenylmethylsulfonyl fluoride (100 µg/ml; Sigma Chemical Co., St. Louis, MO). The cells were then scraped and passed through a 21-gauge needle. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). After 50 µg of the protein were solubilized in sample buffer, the solution was boiled for 5 min before loading onto a 7.5% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane. The specific protein bands were detected with a human Her2/c-neu (Ab-3) monoclonal antibody (Calbiochem, San Diego, CA), E1A (adenovirus type 5) antibody, PARP antibody (BD PharMingen, San Diego, CA), human topoII antibody (TopoGEN, Columbus, OH), and ß-actin monoclonal antibody (Sigma) using the ECL Western blotting analysis system (Amersham) according to the manufacturers’ instructions. Densitometric analysis was performed, and values were normalized with ß-actin loading control.

Flow Cytometry Analysis.
Apoptotic cells were detected by flow cytometry as described previously (24) . TC71 cells were treated as indicated and then were washed with PBS buffer twice; 10⁵ cells were incubated with propidium iodide solution (50 µg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100) for 2 h. Cells were analyzed with a FACScan (Becton Dickinson Co., Mountain View, CA).

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effect of E1A on... Effect of HER2/neu on... Effect of E1A Gene... DISCUSSION REFERENCES

 
High levels of HER2/neu protein were detected in three different Ewing’s sarcoma cell lines (TC7, RD, and A4573; Fig. 1 ) compared with the level in normal human osteoblast cells (NH Osteoblast). SKBR-3, a human breast cancer cell line well known to overexpress HER2/neu, was used as the positive control. Using HER2/neu expression in the normal human osteoblast cells as the baseline, the HER2/neu expression was 20.5-fold higher in TC71 cells, 11.2-fold higher in RD cells, and 13.9-fold higher in A4573 cells. We concluded that all three Ewing’s sarcoma cell lines overexpress HER2/neu.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. HER2/neu expression in three different human Ewing’s sarcoma cell lines. Proteins were extracted from three different human Ewing’s sarcoma cell lines, TC71 (Lane 2), RD (Lane 4), and A4573 (Lane 5); from SKBR-3 human breast cancer cells (Lane 1); and from normal human osteoblast cells (Lane 3) and then subjected to electrophoresis on SDS-polyacrylamide gel. HER2/neu protein (Mr 185,000, top panel) and ß-actin (Mr 42,000, bottom panel) were detected using the human HER2/neu (Ab-3) monoclonal antibody and ß-actin antibody. Densitometric analysis was performed for each band, and values were normalized with a ß-actin loading control. HER2/neu expression in normal human osteoblast cells was used as the baseline. The relative increase in the expression of HER2/neu for each cell line was calculated in comparison with that of normal human osteoblast cells.

	Effect of E1A on HER2/neu Expression.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effect of E1A on... Effect of HER2/neu on... Effect of E1A Gene... DISCUSSION REFERENCES

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Ad-E1A down-regulated HER2/neu expression. TC71 cells were treated with Ad-E1A (Lane 2), Ad-ß-gal (Lane 3), or PBS as control (Lane 1) for 48 h. Proteins were extracted and subjected to electrophoresis on SDS-polyacrylamide gel. Specific antibodies were used for detection of HER2/neu (Mr 185,000), E1A (Mr 46,000), and ß-actin (Mr 42,000) protein expression. Densitometric analysis was performed and adjusted by a ß-actin loading control. The relative level of expression of HER2/neu is expressed in comparison to that in TC71 control cells.

	Effect of HER2/neu on Cell Growth and Sensitivity to Chemotherapeutic Agents.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effect of E1A on... Effect of HER2/neu on... Effect of E1A Gene... DISCUSSION REFERENCES

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Down-regulation of HER2/neu expression by E1A inhibited cell growth. TC71 cells and normal human osteoblast cells were incubated with Ad-E1A, Ad-ß-gal, or PBS as control for 48 h. MTT assays were then performed, and cytostasis was quantified. The results were calculated as percentages of cytostasis compared with levels in control cells. The values were averaged from three different experiments. Bars, SD.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Effects of Ad-E1A on cell sensitivity to VP-16. TC71 cells were incubated with Ad-E1A, Ad-ß-gal, or PBS for 48 h and then treated with different doses of VP-16. Each result was calculated as a percentage of cytostasis compared with that detected in control cells using the MTT assay. The values are averaged from three different experiments. The IC50 is calculated as the dose that inhibited 50% of cell growth. Bars, SD.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Ad-E1A increased the apoptosis induced by VP-16. TC71 cells were incubated with Ad-E1A, Ad-ß-gal, or PBS for 48 h and then treated with 0.3 µM VP-16. Cells were washed with cold PBS and incubated in the buffer; the percentage of cells undergoing apoptosis was quantified by using flow cytometry.

View larger version (52K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Apoptosis of TC71 cells by PARP cleavage. TC71 cells were incubated with Ad-E1A, Ad-ß-gal, or PBS for 48 h and then treated with VP-16. Proteins were extracted and subjected to electrophoresis on SDS-polyacrylamide gel. Anti-PARP monoclonal antibody was used to detect the intact (Mr 116,000) and cleaved (Mr 85,000) PARP forms.

	Effect of E1A Gene Transfer on topoII.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effect of E1A on... Effect of HER2/neu on... Effect of E1A Gene... DISCUSSION REFERENCES

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Ad-E1A increased topoII protein expression. TC71 cells were incubated with Ad-E1A (Lane 2), Ad-ß-gal (Lane 3), or PBS (Lane 1) for 48 h. Proteins were extracted and subjected to electrophoresis on SDS-polyacrylamide gel. Specific antibodies were used to detect topoII (Mr 170,000) and ß-actin (Mr 42,000). Densitometric analysis was performed and adjusted by ß-actin loading control. The relative fold of topoII protein level is expressed in comparison with that in TC71 control cells.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effect of E1A on... Effect of HER2/neu on... Effect of E1A Gene... DISCUSSION REFERENCES

Increased cell sensitivity to chemotherapy after E1A gene transfer has been demonstrated in several different tumor cell lines (27) . However, the mechanism by which E1A enhances drug activity is not clear. E1A indirectly suppresses the HER2/neu promoter through the co-activator p300 (28) . How this function then impacts on drug sensitivity and why the activity of certain drugs is enhanced whereas the activity of others is not are poorly defined. In this study, we show that, in addition to down-regulating HER2/neu, E1A transduction also significantly increases topoII protein expression. Using a specific human topoII antibody, we were able to detect a significant increase in the topoII protein levels in cells transduced with the E1A gene but not in those transduced with the ß-gal gene. Sensitivity to VP-16 and Adriamycin has been shown to be directly linked to the amount of topoII protein in the cell. Indeed, we demonstrated recently that cellular resistance to VP-16 and doxorubicin could be circumvented after human topoII gene transfer (29) . topoII gene transfer into MDA-VP etoposide-resistant human breast cancer cells using an adenoviral vector increased the level of topoII protein, and the sensitivity of these cells to VP-16 and doxorubicin was enhanced. topoII is the sole enzyme target of VP-16 and one of the targets of Adriamycin. In contrast, the mechanism by which cisplatin kills tumor cells has nothing to do with topoII. Thus, our data showing that E1A gene transfer enhanced the activity of VP-16 and Adriamycin, but not that of cisplatin, support our hypothesis that the mechanism by which E1A enhanced the chemosensitivity of Ewing’s sarcoma cells was an increase in topoII.

Ewing’s sarcoma is a relatively rare disease with limited therapeutic options. The majority of patients are initially responsive to chemotherapy with vincristine, Adriamycin, and cyclophosphamide. However, relapsed disease is usually explosive and extremely difficult to treat because of its resistance to chemotherapy. The role of oncogenes and growth factors as well as the etiology of chemoresistance in this disease is poorly understood.

We have demonstrated that HER2/neu is overexpressed in three different human Ewing’s sarcoma cell lines. E1A down-regulated HER2/neu expression, inhibited tumor cell growth, and increased cellular sensitivity to two topoII-directed anticancer drugs. Interestingly, E1A gene transfer had no affect on the growth rate of normal osteoblast cells or on their susceptibility to chemotherapy (Fig. 3 ; other data not shown). These data indicate tumor-specific treatment effect, an important consideration for therapeutic potential. These results suggest that E1A gene therapy may provide a new approach for treatment of Ewing’s sarcoma. Clinical trials with E1A gene therapy are being conducted in patients with metastatic breast cancer and ovarian cancer (30) . Because there is a dearth of therapeutic options for patients with relapsed Ewing’s sarcoma, our data indicate that E1A gene therapy may have potential in the treatment of this disease.

	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 in part by Grant CA82606 (to E.S.K.), Grant CA16672 Cancer Center Support Core Grant (to The University of Texas M.D. Anderson Cancer Center), and a grant from the Kayton Fund.

² To whom requests for reprints should be addressed, at Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 173, Houston, TX 77030. Phone: (713)792-8110, Fax: (713)792-8747.

³ The abbreviations used are: E1A, adenovirus type 5 early region 1A; ß-gal, ß-galactosidase; VP-16, etoposide; topoII, topoisomerase II; PARP, poly(ADP-ribose) polymerase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Received 10/25/00. Accepted 2/13/01.

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