Selenium containing glutathione peroxidase functions in the cellular protection of potentially toxic substances, including peroxides. Nucleotide sequence analysis of cDNAs for this enzyme from human, mouse, rat and bovine sources indicate the presence of an in phase UGA stop codon within the coding sequences. The amino acid corresponding to this stop codon in glutathione peroxidase is always selenocysteine. The biosynthesis and subsequent incorporation of selenocysteine is mediated by a highly unusual, naturally occurring tRNA which is present in all animal species. Recent evidence suggests that the biologically significant form of this tRNA is a post-transcriptionally edited version of the tRNA molecule. It is the objective of this proposal to experimentally establish that editing does occur and study this process. The biological consequences of this phenomenon will be assessed in MCF-7 human cells and their adriamycin-resistant derivative, MCF-7adr. MCF-7adr exhibits elevated levels of both the mRNA for glutathione peroxidase and the presumably edited form of the suppressor tRNA. Introduction of a construct containing the cDNA for glutathione peroxidase under the influence of a strong retroviral promotor into MCF-7 cells results in elevated levels of glutathione peroxidase mRNA but only a marginal increase in enzyme activity. These results are consistent with translational control of glutathione peroxidase and provide a model system to study the incorporation of selenocysteine at UGA codons. The understanding of the regulation of glutathione peroxidase will have implications to clinical chemotherapy as many anti-tumor agents function by the generation of radical species which are substrates for detoxification by this enzyme.
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