Regulation of p53 by metal ions and by antioxidants: dithiocarbamate down-regulates p53 DNA-binding activity by increasing the intracellular level of copper.
Mutations in the p53 tumor suppressor gene frequently fall within the specific DNA-binding domain and prevent the molecule from transactivating normal targets. DNA-binding activity is regulated in vitro by metal ions and by redox conditions, but whether these factors also regulate p53 in vivo is unclear. To address this question, we have analyzed the effect of pyrrolidine dithiocarbamate (PDTC) on p53 DNA-binding activity in cell lines expressing wild-type p53. PDTC is commonly regarded as an antioxidant, but it can also bind and transport external copper ions into cells and thus exert either pro- or antioxidant effects in different situations. We report that PDTC, but not N-acetyl-L-cysteine, down-regulated the specific DNA-binding activity of p53. Loss of DNA binding correlated with disruption of the immunologically "wild-type" p53 conformation. Using different chelators to interfere with copper transport by PDTC, we found that bathocuproinedisulfonic acid (BCS), a non-cell-permeable chelator of Cu1+, prevented both copper import and p53 down-regulation. In contrast, 1,10-orthophenanthroline, a cell-permeable chelator of Cu2+, promoted the redox activity of copper and up-regulated p53 DNA-binding activity through a DNA damage-dependent pathway. We have previously reported that p53 protein binds copper in vitro in the form of Cu1+ (P. Hainaut, N. Rolley, M. Davies, and J. Milner, Oncogene 10:27-32, 1995). The data reported here indicate that intracellular levels and redox activity of copper are critical for p53 protein conformation and DNA-binding activity and suggest that copper ions may participate in the physiological control of p53 function.
Verhaegh
G W
GW
Unit of Mechanisms of Carcinogenesis, International Agency for Research on Cancer, Lyon, France.
Richard
M J
MJ
Hainaut
P
P
D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology*; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology*; D003300:Copper / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007364:Intercalating Agents / Q000494:pharmacology; D017136:Ion Transport / Q000187:drug effects; D015227:Lipid Peroxidation; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010618:Phenanthrolines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000502:physiology; D011759:Pyrrolidines / Q000493:pharmacokinetics; D013859:Thiocarbamates / Q000493:pharmacokinetics / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
D000975:Antioxidants; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D002614:Chelating Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D007364:Intercalating Agents; D010618:Phenanthrolines; D011506:Proteins; D011759:Pyrrolidines; D013859:Thiocarbamates; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C001635:ferroin; C020972:pyrrolidine dithiocarbamic acid; C028559:bathocuproine sulfonate; D003300:Copper; D004247:DNA; D006861:Hydrogen Peroxide; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000111:Acetylcysteine