Poly(ADP-ribose) polymerase-1 regulates the stability of the wild-type p53 protein.
We investigated the interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53 using two different approaches. In the first approach, we used primary and immortalized cells derived from wt and PARP-1 -/- mice. We examined whether PARP-1 deficiency would affect the expression of the wild-type (wt) p53 protein. The inactivation of the PARP-1 gene markedly affected the constitutive expression of the wt p53 protein. Interestingly, only the regularly spliced form of wt p53 was reduced to a barely detectable level in consequence to an approximately 8-fold shortening of its half-life, whereas the level of alternatively spliced p53 remained unchanged. Moreover, reconstitution of cells lacking the PARP-1 gene with the human counterpart restored the normal stability of the regularly spliced p53 protein. In the second approach, we performed experiments with c-Ha-ras transformed primary rat cells overexpressing the p53135val mutant alone or in combination with PARP-1. The advantage of this temperature sensitive p53135val mutant is its oncogenic character at 37 degrees C, connected with cytoplasmic localization of p53, and its tumor suppressor activity at 32 degrees C, accompanied by p53 translocation into the nucleus. No noticeable differences in proliferation and G1 accumulationwere observed between cells expressing p53135val with or without PARP-1. On the other hand, a comparison of the recovery of G1 arrested cells after a shift up to 37 degrees C for both cell lines showed dramatic differences in the kinetics. While cells expressing p53135val rapidly reached the characteristic S-phase level after a shift up to basal temperature, cells additionally expressing PARP-1 rested in G1 despite the temperature elevation. This coincided with exclusively cytoplasmic p53 protein in cells expressing p53135val and predominantly nuclear localization of p53 in p53135val +PARP-1 cells, as evidenced by immunostaining. Determination of the p53 level during the maintenance of cells at 32 degrees C revealed a marked decrease in the level of p53 in cells expressing p53135val alone, whereas in cells coexpressing PARP-1, the level of p53 remained largely unaffected. This indicates that the stability of wild-type p53 greatly differed between both cell lines. Furthermore, the inhibition of PARP-1 activity in G1 arrested cells by 3-aminobenzamide abolished its stabilizing effect on the wild-type p53 protein. Taken together, our results indicate that PARP-1 regulates the stability of the wt p53 protein and that its enzymatic activity is necessary for this stabilizing action.
Wesierska-Gadek
J
J
Institute of Cancer Research, University of Vienna, Austria. Jozefa.Antonia.Gadek-Wesierski@univie.ac.at
Schmid
G
G
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004317:Doxorubicin / Q000494:pharmacology; D005347:Fibroblasts; D006207:Half-Life; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D004317:Doxorubicin; D011065:Poly(ADP-ribose) Polymerases