SAPORIN, A RIBOSOME-INACTIVATING PROTEIN USED TO PREPARE IMMUNOTOXINS, INDUCES CELL-DEATH VIA APOPTOSIS

The plant toxin saporin is a ribosome-inactivating protein which inhib its protein synthesis and growth of both normal and tumour cells. Its cytotoxic activity can be increased by coupling with antibodies recogn izing cell surface antigens. In this work we performed experiments to test the hypothesis that saporin induces cell death via apoptosis. Exp osure to saporin induced apoptosis in different cellular models, such as human peripheral blood B lymphocytes and neutrophils, in the Daudi B-cell line, and in the haemopoietic cell lines HL-60 and TF-1. This w as indicated by: (a) the appearance of typical morphological features such as chromatin condensation, nuclear fragmentation and blebbing of plasma membranes; (b) DNA degradation into oligonucleosomal fragments; (c) the appearance of apoptotic cells on DNA flow cytometry as a cell population with reduced DNA content (A(O) region). The fraction of ce lls showing features of apoptosis ranged from 19 +/- 5% for TF-1 cells to 35 +/- 8% for neutrophils. In experiments with normal peripheral b lood B lymphocytes or with Daudi cells, we compared the activity of na tive saporin with that of an immunotoxin hybrid molecule obtained by b inding the toxin to two bispecific antibodies recognizing both saporin and the B lymphocyte-specific antigen CD22 (Sap/BsAb complexes). Sapo rin bound to the antibodies was 2-3 logs more effective than native sa porin in inducing apoptosis, with maximal inhibitions being observed a t concentrations of 10(-6) M for native saporin and 10(-9)-10(-8) M fo r the hybrid molecules. These findings indicate that treatment with sa porin results in apoptosis of target cells and suggest that this may b e relevant to the therapeutic use of saporin-containing immunotoxins. In fact, if used in vivo as an immunotoxin, its cytotoxic activity cou ld be devoid of more extensive and non-specific tissue damaging effect s as would be the case if saporin induced necrosis of target cells.