Proteasomes are a target of the anti-tumour drug vinblastine

Proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent pathw ay, have a relevant role in many processes crucial for cell physiology and cell cycle progression. Proteasome inhibitors are used to block cell cycle progression and to induce apoptosis in certain cell lines. Here we examine whether proteasomal function is affected by the anti-tumour drug vinblastin e, whose cytostatic action relies mainly on the disruption of mitotic spind le dynamics, The effects of vinblastine on the peptidase activities of huma n 20 S and 26 S proteasomes and on the proteolytic activity of 26 S proteas ome were assessed in the presence of specific fluorogenic peptides and I-12 5-lysozyme-ubiquitin conjugates respectively. The assays of ubiquitin-prote in conjugates and of inhibitory kappaB alpha (I kappaB alpha), Which are ch aracteristic intracellular proteasome substrates, by Western blotting on ly sates from HL60 cells incubated with or without vinblastine, illustrated th e effects of vinblastine on proteasomes in vivo. We also evaluated the effe cts of vinblastine on the signal-induced degradation of I kappaB alpha, Vin blastine at 3-110 muM reversibly inhibited the chymotrypsin-like activity o f the 20 S proteasome and the trypsin-like and peptidyl-glutamyl-peptide hy drolysing activities of both proteasomes, but only at 110 muM vinblastine w as the chymotrypsin-like activity of the 26 S proteasome inhibited; further more, at 25-200 muM the drug inhibited the degradation of ubiquitinated lys ozyme. In HL60 cells exposed for 6h to 0.5-10 muM vinblastine, the drug-dos e-related accumulation of polyubiquitinated proteins, as well as that of a high-molecular-mass form of Ih-Ba, occurred. Moreover. vinblastine impaired the signal-induced degradation of I kappaB alpha. Cell viability throughou t the test was approx. 95 % Proteasomes can be considered to be a new and a dditional vinblastine target.