Article Figures & Data
Figures
- 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 β-actin (Mr 42,000, bottom panel) were detected using the human HER2/neu (Ab-3) monoclonal antibody and β-actin antibody. Densitometric analysis was performed for each band, and values were normalized with a β-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.
- Fig. 2.
Ad-E1A down-regulated HER2/neu expression. TC71 cells were treated with Ad-E1A (Lane 2), Ad-β-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 β-actin (Mr 42,000) protein expression. Densitometric analysis was performed and adjusted by a β-actin loading control. The relative level of expression of HER2/neu is expressed in comparison to that in TC71 control cells.
- 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-β-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.
- Fig. 4.
Effects of Ad-E1A on cell sensitivity to VP-16. TC71 cells were incubated with Ad-E1A, Ad-β-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.
- Fig. 5.
Ad-E1A increased the apoptosis induced by VP-16. TC71 cells were incubated with Ad-E1A, Ad-β-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.
- Fig. 6.
Apoptosis of TC71 cells by PARP cleavage. TC71 cells were incubated with Ad-E1A, Ad-β-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.
- Fig. 7.
Ad-E1A increased topoIIα protein expression. TC71 cells were incubated with Ad-E1A (Lane 2), Ad-β-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 topoIIα (Mr 170,000) and β-actin (Mr 42,000). Densitometric analysis was performed and adjusted by β-actin loading control. The relative fold of topoII α protein level is expressed in comparison with that in TC71 control cells.
Tables
- Table 1
Effect of Ad-E1A on sensitivity of TC71 cells to VP-16, Adriamycin, and cisplatina
Drugs IC50 for drug only IC50 for drug with Ad-E1A Degree of sensitivity enhancement VP-16 1500nm 80nm 18-fold Adriamycin 90nm 18nm 5-fold Cisplatin 630nm 390nm 1.6-fold -
a TC71 cells were incubated with Ad-E1A, Ad-β-gal, or PBS for 48 h and then treated with different doses of VP-16, doxorubicin, or cisplatin. The results were calculated as percentages of cytostasis compared with control cells using the MTT assay. The values are the averages of results from three different experiments. The IC50 is calculated as the dose that inhibited 50% of cell growth. The degree of enhancement was calculated as the IC50 for drug only divided by the IC50 for drug in cells treated with Ad-E1A.
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Volume 61, Issue 8
