Ribosome-inactivating proteins (RIPs) remove a specific adenine from 28S rRNA leading to inactivation of ribosomes and arrest of translation. Great interest as to a possible second physiological substrate for RIPs came from the observation that in vitro RIPs remove adenine from DNA. This paper addresses the problem of nuclear lesions induced by RIPs in human endothelial cells susceptible to the bacterial RIP Shiga toxin 1 and the plant RIP ricin. With both toxins, nuclear DNA damage as evaluated by two independent techniques (alkaline-halo assay and alkaline filter elution) appears early, concomitant with (ricin) or after (Shiga toxin 1) the inhibition of protein synthesis. At this time, the annexin V binding assay, caspase 3 activity, the formation of typical < or = 50 Kb DNA fragments, and changes in morphology associated with apoptosis were negative. Furthermore, a block of translation comparable to that induced by RIPs, but obtained with cycloheximide, did not induce nuclear damage. Such damage is consistent with the enzymatic activity (removal of adenine) of RIPs acting in vitro on RNA-free chromatin and DNA. The results unequivocally indicate that RIPs can damage nuclear DNA in whole cells by means that are not secondary to ribosome inactivation or apoptosis.

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Peroxynitrite stimulates in U937 cells release of arachidonic acid (AA) sensitive to various phospholipase A(2) (PLA(2)) inhibitors, including arachidonyl trifluoromethyl ketone (AACOCF(3)), which specifically inhibits cytosolic PLA(2) (cPLA(2)). This response linearly increases using non toxic concentrations of the oxidant, and reaches a plateau at levels at which toxicity becomes apparent. Three separate lines of evidence are consistent with the notion that AA generated by cPLA(2) promotes survival in cells exposed to peroxynitrite. Firstly, toxicity was suppressed by nanomolar levels of exogenous AA, or by AA generated by the direct PLA(2) activator melittin. Secondly AACOCF(3), or other PLA(2) inhibitors, promoted cell death after exposure to otherwise non toxic concentrations of peroxynitrite; exogenous AA abolished the enhancing effects mediated by the PLA(2) inhibitors. Finally, U937 cells transfected with cPLA(2) antisense oligonucleotides were killed by concentrations of peroxynitrite that were non-toxic for cells transfected with nonsense oligonucleotides. This lethal response was insensitive to AACOCF(3) and prevented by exogenous AA.

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