Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells.
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.
Brigotti
Maurizio
M
Dipartimento di Patologia Sperimentale, Università degli Studi di Bologna, Italy. brigotti@alma.unibo.it
Alfieri
Roberta
R
Sestili
Piero
P
Bonelli
Mara
M
Petronini
Pier Giorgio
PG
Guidarelli
Andrea
A
Barbieri
Luigi
L
Stirpe
Fiorenzo
F
Sperti
Simonetta
S
D000225:Adenine / Q000378:metabolism; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects* / Q000648:ultrastructure; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D053938:DNA Fragmentation; D004727:Endothelium / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D012276:Ricin / Q000633:toxicity*; D022622:Shiga Toxin 1 / Q000633:toxicity*
D002843:Chromatin; D022622:Shiga Toxin 1; D012276:Ricin; D009699:N-Glycosyl Hydrolases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D000225:Adenine