Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis

Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 2 6 S proteasome in regulating apoptosis, although the precise nature of thei r involvement is not known. In the present study, control and Bcl-x(L)-over expressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Ba sal proteasome activity appeared to be approximate to 30 % lower in bcl-x(L ) cells compared with control cells using a substrate for the chymotrypsin- like activity. However, no difference in proteasome activity was detected u sing substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysin g activities. In addition, protein levels of the 20 S proteasome beta-subun it, as determined by Western blot analyses, were similar in control and bcl -x(L) cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, ap optosis in bcl-x(L) cells (22%) was less than that observed in control cell s (34 %). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl-7-amino-4-methylcoumarin (Ac-DEVD-AMC), was twice that observed in bcl-x(L) cells. By 48 h after MG1 32 treatment, apoptosis and caspase activity in bcl-x(L) cells were similar to those observed in control cells at 24 h, Proteasome inhibition stimulat ed increases in hsp70 protein levels in control and bcl-x(L) cells by 12 h, although the maximal increases found in bcl-x(L) cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spect rum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, preve nted MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEV D-aldehyde, afforded less protection, although both inhibitors completely i nhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl- x(L) provide some protection against proteasome inhibitor-induced apoptosis .