Differential susceptibility of normal and PARP knock-out mouse fibroblasts to proteasome inhibitors.
Recently we found a clearly reduced basal level of wt p53 protein in PARP-deficient cells. Interestingly, PARP deficiency affected only regularly spliced (RS) wt p53. No significant difference of the p53 transcription rate was observed between wt and PARP-lacking cells. To clarify whether the reduction of RS p53 protein is due to a lower translation rate or rather to its instability in the absence of functional PARP, we investigated the effect of the inhibition of proteasome activity and nuclear export on the p53 level. The p53 half-life was approximately eight-fold decreased in PARP-lacking cells. Surprisingly, treatment with three proteasome inhibitors increased RS p53 in normal but not in PARP-deficient cells. However, the inhibition of nuclear export resulted in a considerable accumulation of RS p53 in the latter. Therefore, we decided to increase concentrations of the inhibitors. Their higher concentrations strongly affected viability of normal, but not of PARP-deficient cells, about 70% of MEFs died. Interestingly, higher concentrations of proteasome inhibitors resulted in the appearance of RS p53 in PARP-lacking fibroblasts. Reconstitution of PARP-deficient cells with PARP restored the normal susceptibility to proteasome inhibitors thereby unequivocally demonstrating that the enhanced cytotoxicity of proteasome inhibitors and their action on p53 level depends on the presence of functional PARP.
Wesierska-Gadek
J
J
Institute of Cancer Research, University of Vienna, A-1090 Vienna, Austria. Jozefa.Antonia.Gadek-Wesierski@univie.ac.at
Bohrn
E
E
Herceg
Z
Z
Wang
Z Q
ZQ
Wurzer
G
G
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010641:Phenotype; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000235:genetics* / Q000502:physiology*; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis
D015853:Cysteine Proteinase Inhibitors; D005231:Fatty Acids, Unsaturated; D005456:Fluorescent Dyes; D007211:Indoles; D007976:Leupeptins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011506:Proteins; D016159:Tumor Suppressor Protein p53; C090535:benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal; C069861:calpain inhibitor 2; C067713:lactacystin; C007293:DAPI; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine; C038753:leptomycin B