Related Experiment Videos
Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis
J D Robertson1, K Datta, S S Biswal
1Division of Pharmacology, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712-1074, USA.
Abstract:
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.
Insights
Heat-shock protein 70 (hsp70) and Bcl-x(L) protect against apoptosis induced by proteasome inhibitors. Blocking hsp70 potentiated apoptosis, while caspase inhibition prevented it, highlighting their roles in cell survival.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Heat-shock protein 70 (hsp70) and the 26S proteasome are implicated in apoptosis regulation.
- The precise mechanisms of their involvement remain unclear.
- Bcl-x(L) is known to inhibit apoptosis.
Purpose of the Study:
- To investigate the roles of hsp70 and Bcl-x(L) in apoptosis induced by proteasome inhibition.
- To elucidate the relationship between proteasome activity, hsp70, and caspase activation in the context of Bcl-x(L) expression.
Main Methods:
- FL5.12 cell lines overexpressing Bcl-x(L) and control cells were treated with the proteasome inhibitor MG132.
- Proteasome activity was assessed using specific substrates.
- Protein levels of 20S proteasome beta-subunit were analyzed by Western blot.
- Apoptosis and caspase activity were measured.
- hsp70 levels were modulated using antisense oligonucleotides.
- Caspase activity was inhibited using broad-spectrum and specific caspase inhibitors.
Main Results:
- Bcl-x(L) overexpression showed slightly reduced basal chymotrypsin-like proteasome activity but similar overall proteasome activity and 20S proteasome beta-subunit levels compared to controls.
- MG132 treatment induced less apoptosis and lower caspase activity in Bcl-x(L) cells compared to control cells.
- Proteasome inhibition increased hsp70 levels, with lower induction in Bcl-x(L) cells.
- Blocking hsp70 induction potentiated MG132-induced apoptosis.
- Caspase inhibition prevented MG132-induced apoptosis.
Conclusions:
- Both hsp70 and Bcl-x(L) confer protection against proteasome inhibitor-induced apoptosis.
- hsp70 and Bcl-x(L) appear to act through modulating caspase activity.
- These findings highlight the interplay between proteasome function, hsp70, and the Bcl-2 family in cell survival pathways.