p53 protein oxidation in cultured cells in response to pyrrolidine dithiocarbamate: a novel method for relating the amount of p53 oxidation in vivo to the regulation of p53-responsive genes

A novel method was developed to determine the oxidation status of proteins in cultured cells. Methoxy-polyethylene glycolmaleimide MW 2000 (MAL-PEG) w as used to covalently tag p53 protein that was oxidized at cysteine residue s in cultured cells. Treatment of MCF7 breast cancer cells with pyrrolidine dithiocarbamate (PDTC), a metal chelator, resulted in a minimum of 25% oxi dation of p53. The oxidized p53 had an average of one cysteine residue oxid ized per p53 protein molecule. The effect of PDTC treatment on downstream c omponents of the p53 signal-transduction pathway was tested. PDTC treatment prevented actinomycin D-mediated up-regulation of two p53 effector gene pr oducts, murine double minute clone 2 oncoprotein and p21(WAF1/CIP1) (where WAF1 corresponds to wild-type p53-activated fragment 1 and CIP1 corresponds to cyclin-dependent kinase-interacting protein 1). Actinomycin D treatment led to accumulation of p53 protein in the nucleus. However, when cells wer e simultaneously treated with PDTC and actinomycin D, p53 accumulated in bo th the nucleus and the cytoplasm. The data indicate that an average of one cysteine residue per p53 protein molecule is highly sensitive to oxidation and that p53 can be efficiently oxidized by PDTC in cultured cells. PDTC-me diated oxidation of p53 correlates with altered p53 subcellular localizatio n and reduced activation of p53 downstream effector genes. The novel method for detecting protein oxidation detailed in the present study may be used to determine the oxidation status of specific proteins in cells.