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Immunotoxin-Induced Apoptosis

R&D Systems, Inc., Minneapolis, Minnesota, USA

J.P. Houchins, Ph.D, R&D Systems, Inc., 614 McKinley Place NE, Minneapolis, Minnesota 55413, USA. Telephone: 612-379-2956; Fax: 612-379-6580; e-mail:info{at}rndsystems.comWebsite: http://www.rndsystems.com

Immunotoxins are cytotoxic agents usually assembled as recombinant fusion proteins composed of a targeting domain and a toxin. The targeting domain controls the specificity of action and is usually derived from an antibody Fv fragment, a growth factor, or a soluble receptor. The protein toxins are obtained from bacteria (e.g., Pseudomonas endotoxin [PE] or diptheria toxin [DT]) or from plants (e.g., ricin). Immunotoxins have been studied as treatments for cancer, graft-versus-host disease, autoimmune diseases, and AIDS [1].

The bacterial toxins PE and DT act via the ADP-ribosylation of elongation factor [2], thereby inactivating it [1]. This results in the arrest of protein synthesis and subsequent cell death. These toxins can also induce apoptosis, although the mechanism is unknown [2-4]. Insight into the mechanism was obtained from a study of a PE-based immunotoxin directed against Lewis^y antigen, which is expressed on many epithelial cancers including colon and breast [5]. The effect of toxin on cell morphology and common biochemical events that occur during apoptosis were reported.

Two common features of apoptotic cell death are the activation of a group of cysteine proteases called caspases and the caspase-catalyzed cleavage of so-called "death substrates" such as the nuclear repair enzyme poly(ADP-ribose) polymerase (PARP) [6]. Exposure of a breast cancer cell line to the PE immunotoxin caused a fivefold increase in caspase activity and induced PARP cleavage [5]. Results with caspase inhibitors and fluorescent protease substrates bearing specific recognition sequences suggest that the toxin activates caspase-3 or a closely related protease.

Immunotoxin-induced cytotoxicity was compared with that of TNF-, which is totally dependent on apoptotic mechanisms for cell death [5]. Addition of a caspase inhibitor or overexpression of the anti-apoptotic protein Bcl-2 completely shut down the apoptotic pathway. Neither PE nor TNF triggered PARP cleavage under these circumstances, and TNF-induced cytotoxicity was eliminated. PE-induced cytotoxicity still occurred through its effect on protein synthesis, albeit with two- to threefold-reduced sensitivity.

It is clear from these results that PE kills cells by two different mechanisms: inhibition of protein synthesis and induction of apoptosis through caspase activation. The relationship between these two mechanisms is unclear, however, apoptosis is not simply a byproduct of the arrest of protein synthesis. Cycloheximide, which blocks protein synthesis by a different mechanism, did not induce cleavage of PARP [5].

PE-induced cytotoxicity is only partially dependent on an apoptotic mechanism. The contribution of the apoptotic pathway, however, may be significant under conditions of limiting concentration or limited duration of exposure to an immunotoxin.

View larger version (20K): [in this window] [in a new window]   Figure 1 . The immunotoxin, consisting of a targeting domain and a PE toxin, binds to the cell surface and is internalized by receptor-mediated endocytosis. Proteolytic cleavage within the trans-golgi network separates the toxin from the targeting domain. The toxin is then released into the cytosol where it acts to inactivate elongation factor 2 (EF-2) and activate caspase- 3 by an unknown mechanism. Caspase-3 catalyzes cleavage of PARP.

1. Thrush GR, Lark LR, Clinchy BC et al. Immunotoxins: an update. Annu Rev Immunol 1996;14:49-71.[CrossRef][Medline]

2. Chang MP, Bramhall J, Graves S et al. Internucleosomal DNA cleavage precedes diptheria toxin-induced cytolysis. Evidence that cell lysis is not a simple consequesnce of translation inhibition. J Biol Chem 1989;264:15261-15267.[Abstract/Free Full Text]

3. Morimoto H, Bonavida B. Diptheria toxin-and Pseudomonas A toxin-mediated apoptosis. ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha. J Immunol 1992;149:2089-2094.[Abstract]

4. Brinkmann U, Brinkmann E, Gallo M et al. Role of CAS, a human homologue to the yeast chromosome segregation gene CSE1, in toxin and tumor necrosis factor mediated apoptosis. Biochemistry 1996;35:6891-6899.[CrossRef][Medline]

5. Keppler-Hafkemger A, Brinkmann U, Pastan I. Role of caspases in immunotoxin-induced apoptosis of cancer cells. Biochemistry 1998;37:16934-16942.[CrossRef][Medline]

6. Nicholson DW, Thornberry NA. Caspases: killer proteases. Trends Biochem Sci 1997;22:229-306.

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