Altering Cell Survival by Modulating Levels of Mitochondrial DNA Repair Enzymes

摘要

Efficient repair of oxidative damage in mtDNA is essential for survival of the cells undergoing oxidative stress, and may play a role in cancer cells resistance to a radiation therapy. Decreasing mtDNA repair capacity in cancer cells can make them more vulnerable to cancer therapy. Our previous results demonstrated that stable expression of E.coli Exonuclease III in mitochondria of breast cancer cells diminishes mtDNA repair capacity following oxidative stress, which leads to a decrease in long-term cell survival. Because of the temporary nature of cancer therapy, only transient introduction of proteins into cells is required. In this study we utilize the novel method for direct delivery of purified proteins into cells via protein transduction, combined, for the first time, with targeting of the transduced proteins to mitochondria. Recombinant EGFP and Exonuclease III bearing the mitochondrial targeting signal from human Manganese Superoxide Dismutase and protein transduction domain from HIV-1 Tat protein were expressed in E.coli and purified. Our results show the high efficiency of the transduction as well as mitochondrial localization of the transduced proteins. Current studies are under way to assess the effect of the protein transduction on Exonuclease III activity in the mitochondria and its consequences for mtDNA repair and cell survival.