REGULATION OF P53 BY METAL-IONS AND BY ANTIOXIDANTS - DITHIOCARBAMATEDOWN-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 transactivat ing normal targets, DNA-binding activity is regulated in vitro by meta l ions and by redox conditions, but whether these factors also regulat e p53 in vivo is unclear, To address this question, we have analyzed t he effect of pyrrolidine dithiocarbamate (PDTC) on p53 DNA-binding act ivity in cell lines expressing wild-type p53. PDTC is commonly regarde d as an antioxidant, but it can also hind and transport external coppe r 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 coppe r transport by PDTC, we found that bathocuproinedisulfonic acid (BCS), a non-cell-permeable chelator of Cu1+, prevented both copper import a nd 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 p athway, We have previously reported that p53 protein binds copper in v itro in the form of Cu1+ (P. Hainaut, N. Rolley, M. Davies, and J. Mil ner, Oncogene 10:27-32, 1995). The data reported here indicate that in tracellular levels and redox activity of copper are critical for p53 p rotein conformation and DNA-binding activity and suggest that copper i ons may participate in the physiological control of p53 function.