Ribosome-inactivating proteins (RIP) represent a family of plant and bacterial toxins that inhibit protein synthesis and are cytotoxic to mammalian cells. Ricin is isolated from seeds of the castor bean plant and has potential as a weapon of bioterrorism. Shiga-like toxins (stx) are produced from enterohemorrahagic Escherichia coli strains, and contamination of food by stx represents a substantial public health threat. Both ricin and stx consist of A and B chains. The A-chain of each protein inactivates the ribosome by cleaving an adenine from the ribosomal RNA. The B-chain of ricin (RTB) binds to galactose binding sites on the cell surface while the pentameric B subunit of stx binds to the cell surface receptor, globotriaosylceramide to facilitate cell entry. While both ricin and stx inhibit protein synthesis and induce cell death, the molecular mechanisms that underlie these effects are relatively unexplored. An ultimate goal of this research is to produce recombinant mutant ricin A-chain (RTA) proteins to study the role of protein synthesis inhibition in ricin-induced cytotoxicity. Therefore, we were interested in establishing a mammalian cell culture model that was sensitive to RTA alone. The cell line MAC-T, an immortalized, nontransformed epithelial cell line, was more sensitive than Vero cells and HeLa cells in terms of the time it took to induce caspase-3/7 activation. While ricin induced higher caspase-3/7 activity than RTA at lower concentrations (0.1 to 10 ng/ml), similar caspase activation was observed at concentrations of 0.1 µg/ml with either protein. Ribosome depurination, protein synthesis inhibition, and apoptosis were observed in MAC-T cells treated with RTA alone. RTA alone also induced JNK and p38 MAP kinase activation in a time- and concentration-dependent manner that preceded apoptosis. Inhibition of the JNK pathway by chemical inhibitors or small interfering RNA reduced RTA-induced apoptosis. In contrast, inhibition of the p38 MAP kinase pathway had little effect on RTA-induced apoptosis.

In summary, the major findings of this research are the establishment of the MAC-T cell line as a sensitive cell culture model for future study of ricin and the finding that the JNK pathway plays a major role in RTA-induced cytotoxicity.