Role of caspases in immunotoxin-induced apoptosis of cancer cells

Immunotoxins composed of antibodies linked to plant or bacterial toxins are being evaluated in the treatment of cancer. It is known that the toxin moi eties of immunotoxins, including Pseudomonas exotoxin A (PE), diphtheria to xin, and ricin, are capable of inducing apoptosis. Since the efficiency of induction of apoptosis and the apoptosis pathway may have direct effects on the therapeutic usefulness of immunotoxins, we have studied how B3(Fv)-PE3 8, a genetically engineered immunotoxin in which the Fv fragment of an anti body is fused to a mutated form of PE, induces apoptosis of the MCF-7 breas t cancer cell line. We show for the first time that a PE-containing immunot oxin activates ICE/ced-3 proteases, now termed caspases, and causes charact eristic cleavage of the "death substrate" poly(ADP)-ribose polymerase (PARP ) to an 89 kDa fragment with a time course of cleavage comparable to that i nduced by TNF alpha. Also the fluorescent substrate? DEVD-AFC, is cleaved 2 -4-fold more rapidly by lysates from B3(Fv)-PE38 treated MCF-7 cells than u ntreated control cells, suggesting that a CPP32-like caspase is involved in B3(Fv)-PE38-mediated apoptosis. B3(Fv)-PE38 -induced PARP cleavage is inhi bited by several protease inhibitors known to inhibit caspases (zVAD-fmk, z DEVD-fmk, zIETD-fmk) as well as by overexpression of Bcl-2 providing additi onal evidence for caspase involvement. zVAD-fmk, a broad spectrum inhibitor of most mammalian caspases, prevents the early morphological changes and l oss of cell membrane integrity produced by B3(Fv)-PE38, but not its ability to inhibit protein synthesis, arrest cell growth, and subsequently kill ce lls. Despite inhibition of apoptosis, the immunotoxin is still capable of s elective cell killing, which indicates that B3(Fv)-PE38 kills cells by two mechanisms: one requires caspase activation, and the other is due to the ar rest of protein synthesis caused by inactivation of elongation factor 2. Th e fact that an immunotoxin can specifically kill tumor cells without the ne ed of inducing apoptosis makes such agents especially valuable for the trea tment of cancers that are protected against apoptosis, e.g., by overexpress ion of Bcl-2.