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 c ancer. In contrast to mutant p53, wild-type (wt) p53 protein is presen t at low levels due to rapid degradation by proteasome. We demonstrate d 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 a brogation of ubiquitination, Consistent with this, the E6 oncoprotein which targets p53 for ubiquitination abolished stabilization of p53 pr otein by DNA-damaging drugs but not by proteasome inhibitors. In contr ast to the effects on wt p53, inhibitors of proteolysis downregulated mutant p53. Regulation of p53 levels can be explained by a feedback me chanism where wt p53 transcriptionally induces ''sensor'' proteins (Md m-2, as an example) and these, in turn, target p53 for degradation. Li ke p53, Mdm-2 is degraded by proteasome. Therefore, inhibition of prot easome 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 inh ibition of Mdm-2 synthesis. An inhibitor of transcription, alpha-amani tin, dramatically induced wt p53 protein, whereas Mdm-2 protein was do wnregulated. Moreover, alpha-amanitin increased p53 protein levels in E6-transfected cells. Although inhibitors of transcription, such as ac tinomycin D, also damage DNA, reduction of Mdm-2 or other putative ''s ensor'' proteins may contribute to their p53-stabilizing activity, Sim ilarly, antimetabolites augment accumulation of wt p53 due to interfer ence with RNA synthesis. (C) 1998 Academic Press.