Thioltransferase, also known as glutaredoxin is an enzyme that catalyzes dithiol-disulfide exchange reactions and participates in a ribonucleotide reductase electron transport pathway. Recent studies in the PIs laboratory have identified a new function for thioltransferase, namely dehydroascorbate reductase. The regeneration of ascorbate from dehydroascorbate requires glutathione as reductant, thus closely linking ascorbate with glutathione intracellularly. Previous findings that transformed or malignant cells of various origins have significantly decreased thioltransferase activity compared with normal parental cell lines suggested that these tumor cells would be exceptionally vulnerable to natural or drug-induced oxidative cytotoxicity mechanisms. In the absence of normal levels of thioltransferase and the presence of ascorbate and Adriamycin, normal (SL88) and transformed human fibroblasts (HT 1080), drug sensitive wild-type (WT) and resistant (AdrR) MCF-7 human breast cell lines and high and low metastatic potential MDA-MB-435 breast cell tumor cells will be investigated for cytotoxicity from Adriamycin in the presence and absence of ascorbic acid. Human thioltransferase cDNA will be cloned, sequenced and expressed for use in antibody production against thioltransferase expression. Thioltransferase cDNA will be used to measure mRNA abundance by Northern blot analysis in efforts to understand the regulation of thioltransferase transcription. Transection of human thioltransferase cDNA into Adriamycin sensitive WT MCF-7 human breast tumor cells will be carried out to determine whether increased thioltransferase expression is directly related to Adriamycin resistance in MCF-7 cells. Other cell lines sensitive and resistant to cis-platin will be studied for thioltransferase and other glutathione related enzyme activities based on the known potent inhibition of thioltransferase by the platinum complex drugs. Another strong thioltransferase inhibitor was found to be methylglyoxal. Further inhibition studies of this long-known antitumor agent will be undertaken. These studies are designed to provide new information on the role of thioltransferase in normal and malignant cells with respect to its interrelationship between glutathione and ascorbic acid metabolism.
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