Inhibitors of transcription, proteasome inhibitors, and DNA-damaging drugs differentially affect feedback of p53 degradation.
Mutations of the p53 gene are the most common abnormalities in human cancer. In contrast to mutant p53, wild-type (wt) p53 protein is present at low levels due to rapid degradation by proteasome. We demonstrated that wt p53 protein stabilization following DNA damage or proteasome inhibition did not abolish the wild-type conformation. DNA damage did not cause accumulation of ubiquitinated forms of wt p53, suggesting abrogation of ubiquitination. Consistent with this, the E6 oncoprotein which targets p53 for ubiquitination abolished stabilization of p53 protein by DNA-damaging drugs but not by proteasome inhibitors. In contrast to the effects on wt p53, inhibitors of proteolysis downregulated mutant p53. Regulation of p53 levels can be explained by a feedback mechanism where wt p53 transcriptionally induces "sensor" proteins (Mdm-2, as an example) and these, in turn, target p53 for degradation. Like p53, Mdm-2 is degraded by proteasome. Therefore, inhibition of proteasome caused accumulation of Mdm-2, leading to degradation of mutant p53 by the remaining proteolytic activity of the cell. We propose that inhibition of transcription should increase wt p53 protein due to inhibition of Mdm-2 synthesis. An inhibitor of transcription, alpha-amanitin, dramatically induced wt p53 protein, whereas Mdm-2 protein was downregulated. Moreover, alpha-amanitin increased p53 protein levels in E6-transfected cells. Although inhibitors of transcription, such as actinomycin D, also damage DNA, reduction of Mdm-2 or other putative "sensor" proteins may contribute to their p53-stabilizing activity. Similarly, antimetabolites augment accumulation of wt p53 due to interference with RNA synthesis.
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Medicine Branch, National Cancer Institute, NIH, Bethesda, Maryland, 20892, USA.
Chuman
Y
Y
Fojo
T
T
Blagosklonny
M V
MV
D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D005246:Feedback / Q000187:drug effects / Q000502:physiology; D005260:Female; D006801:Humans; D008297:Male; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D004273:DNA, Neoplasm; D009097:Multienzyme Complexes; D016159:Tumor Suppressor Protein p53; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex