Figure 3. Overexpression of human ASMase via Ad5H2E-PPE1(3x)-ASMase increases lysosomal and secretory ASMase activity, radiosensitizes BAEC, and attenuates bFGF protection against radiation-induced apoptosis.

(A) Cellular homogenates and serum-free conditioned media were harvested from BAEC transduced with Ad5Empty or Ad5H2E-PPE1(3x)-ASMase and assayed for ASMase activity using [¹⁴C-methylcholine]sphingomyelin as substrate in the presence of 1 mM EDTA (cellular homogenates) or 0.1 mM Zn²⁺ (conditioned media). (B) BAEC transduced with Ad5Empty or Ad5H2E-PPE1(3x)-ASMase were pre-treated with 1 ng/ml bFGF 15 minutes prior to 10 Gy irradiation. Apoptosis was assessed at various time points after IR by morphologic analysis following bis-benzimide staining. Data (mean ± SEM) are collated from 3 experiments performed in triplicate in which 400 nuclei were analyzed per sample.