Cell killing effect of heparin-binding EGF-like growth factor Pseudomonas exotoxin on human hepatoma cells

Heparin-binding, EGF-like growth factor (HB-EGF) is a potent mitogen for smooth muscle cell, fibroblast, and it also stimulates hepatocyte proliferation. We generated several chimeric toxins by fusing the cDNA sequence of HB-EGF and the mutant of Pseudomonas exotoxin, PE^4EKDEL (PE) that lacks the binding ability to a specific receptor. HB-EGF-PE was generated by fusing the DNA fragment encoding the full length mature HB-EGF polypeptide to the N-terminus of PE^4EKDEL, while HB-PE was generated by fusing the 45 N-terminal heparin-binding sequence to PE^4EKDEL. HB-EGF-PE was capable of binding both to the EGF receptor and heparin sulfate proteoglycans (HSPGs), whereas HB-PE was capable of binding only to HSPGs on the target cells. Human hepatoma cells, SK-Hepl, Hep-G2 and PLC/PRF/5 were killed in a very low concentration, of HB-EGF-PE with the ID₅₀ of 0.1–0.5 ng/ml. HB-PE could also kill SK-Hepl with the ID₅₀ of 50 ng/ml, whereas it was resistant to PE. Both exogenous EGF and heparin inhibited the cytotoxicity of HB-EGF-PE. These results indicated the existence of two alternative pathways for the internalization of the chimeric toxins defined by two different targets, EGFR and HSPGs.