Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis.
Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-x(L)-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be approximately 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 using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome beta-subunit, 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, apoptosis in bcl-x(L) cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-7-amino-4-methylcou marin (Ac-DEVD-AMC), was twice that observed in bcl-x(L) cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-x(L) cells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated 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-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-x(L) provide some protection against proteasome inhibitor-induced apoptosis.
Robertson
J D
JD
Division of Pharmacology, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712-1074, USA.
Datta
K
K
Biswal
S S
SS
Kehrer
J P
JP
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D001402:B-Lymphocytes; D002172:Canavanine / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000187:drug effects*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004357:Drug Synergism; D004789:Enzyme Activation; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006412:Hematopoietic Stem Cells; D007377:Interleukin-3 / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D009097:Multienzyme Complexes / Q000187:drug effects*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis*; D051020:bcl-X Protein
C494812:Bcl2l1 protein, mouse; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D018840:HSP70 Heat-Shock Proteins; D007377:Interleukin-3; D007976:Leupeptins; D009097:Multienzyme Complexes; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; C067713:lactacystin; D002172:Canavanine; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine