Mild hyperthermia plus adenoviral p53 over-expression additively inhibits the viability of human malignant glioma cells

Figures & data

Figure 1. (a) Treatment of malignant glioma cells with adenoviral p53 gene transfer. (A) U373-MG, (B) U251-MG and (C) D54 glioma cells were treated with Ad5CMV-GFP or Ad5CMV-p53 at the MOI indicated on the X-axis for 72 h and an MTT assay was performed. (b) Treatment with adenoviral p53 over-expression, heat shock or their combination. (A) U373-MG, (B) U251-MG and (C) D54 glioma cells were treated with Ad5CMV-GFP or Ad5CMV-p53 for 48 h, heat-treated at 37 or 43°C for 2 and 48 h later an MTT assay was performed.

Figure 2. Cell cycle analysis of (a) U373-MG, (b) U251-MG and (c) D54 glioma cells after p53 over-expression, heat-shock treatment or their combination. (a) No treatment, (b) heat-shock treatment at 43°C for 2 h only, (c) Ad5CMV-GFP only, (d) Ad5CMV-GFP + heat-shock treatment at 43°C for 2 h, (e) Ad5CMV-p53 only and (f) Ad5CMV-p53 + heat-shock treatment at 43°C for 2 h.

Figure 3. Flow cytometric analysis of (a) U373-MG, (b) U251-MG and (c) D54 glioma cells with TUNEL staining for detection of DNA fragmentation. (a) No treatment, (b) heat-shock treatment at 43°C for 2 h only, (c) Ad5CMV-GFP only, (d) Ad5CMV-GFP + heat-shock treatment at 43°C for 2 h, (e) Ad5CMV-p53 only, (f) Ad5CMV-p53 + heat-shock treatment at 43°C for 2 h and (g) treatment with DNase I as a positive control.

Figure 4. Caspase inhibition as shown by an MTT assay performed in (a) U373-MG, (b) U251-MG and (c) D54 glioma cells using the pan-caspase inhibitor zVAD-fmk along with adenoviral p53 gene transfer and/or heat shock at 43°C.

Figure 5. Immunoblotting assay for the expression of p21 protein after p53 over-expression, heat-shock treatment or their combination. Cell lysates were prepared from (a) U373-MG, (b) U251-MG and (c) D54 cells, which were treated with (a) Ad5CMV-GFP only, (b) Ad5CMV-GFP + heat-shock treatment at 43°C for 2 h, (c) Ad5CMV-p53 only and (d) Ad5CMV-p53 + heat-shock treatment at 43°C for 2 h. Proteins were subjected to immunoblot analysis using anti-p21 antibody. Anti-β-actin antibody was used to confirm equivalent protein loading.