REDIRECTION OF REDOX PROTEINS TO THE NUCLEUS OF EUKARYOTIC CELLS

点击翻译按钮获取中文摘要

Active oxygen species are implicated in ionizing radiation's and some antineoplastic drugs' cytotoxic mechanism of action. We already have shown that cells engineered to overexpress glutathione peroxidase (a protein known to detoxify hydrogen peroxide and alkylperoxides) are no more resistant to adriamycin or radiation induced cytotoxicity than are control cells. The lack of effect can be explained by the fact that glutathione peroxidase, although overexpressed, is localized normally in the cytoplasmic compartment and the primary target of the two damaging agents is DNA within the nuclear compartment. Therefore, glutathione peroxidase gene has been re-engineered by site directed mutagenesis to localize to the nucleus. Cooper/zinc dependent superoxide dismutase (detoxifies superoxide anion radical) and catalase (detoxifies hydrogen peroxide) genes have also been engineered to traffic to the nucleus. In the case of catalase, the peroxisome directing signal had to be removed from the gene and replaced by a nuclear targeting signal. Eukaryotic cells are being transformed with vectors containing the engineered constructs to test if active oxygen species are: l) responsible for cytotoxicity and 2) can be detoxified within the nuclear compartment. The reengineered proteins were chosen to provide information of which active oxygen species is promoting cell death. The projects require mRNA isolation, RT/PCR to isolate the respective gene of interest, SDM for redirection of the expressed proteins, tissue culture and transfection vector and regulatable promoter containing plasmid construction. This project has the potential to provide greater understanding of the mechanism of action of antineoplastic drugs and radiation.