DNA Repair 2 (2003) 471–482 The expression of Exonuclease III from E. coli in mitochondria of breast cancer cells diminishes mitochondrial DNA repair capacity and cell survival after oxidative stress Inna N. Shokolenko a , Mikhail F. Alexeyev b , Fredika M. Robertson c , Susan P. LeDoux a , Glenn L. Wilson a,∗ a Departments of Cell Biology and Neuroscience, University of South Alabama College of Medicine, Mobile, AL 36688, USA b Pharmacology, University of South Alabama College of Medicine, Mobile, AL 36688, USA c Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University College of Medicine & Public Health, Columbus, OH 43210, USA Accepted 17 December 2002 Abstract The ability to sensitize cancer cells to radiation would be highly beneficial for successful cancer treatment. One mode of action for ionizing radiation is the induction of cell death through infliction of extensive oxidative damage to cellular DNA, including mitochondrial DNA (mtDNA). The ability of cells to repair mtDNA and otherwise maintain the integrity of their mitochondria is vital for protection of the cells against oxidative damage. Because efficient repair of oxidative damage in mtDNA may play a crucial role in cancer cell resistance, interference with this repair process could be an effective way to achieve a radiation sensitive phenotype in otherwise resistant cancer cells. Successful repair of DNA is achieved through a precise and highly regulated multistep process. Expression of excessive amounts of one of the repair enzymes may cause an imbalance of the whole repair system and lead to the loss of repair efficiency. To study the effects of changing mtDNA repair capacity on overall cell survival following oxidative stress, we expressed a bacterial repair enzyme, Exonuclease III (ExoIII) containing the mitochondrial targeting signal of manganese superoxide dismutase, in a human malignant breast epithelial cell line, MDA-MB-231. Following transfection, specific exonuclease activity was found in mitochondrial extracts. In order to examine the effects on repair of oxidative damage in mtDNA, cells were exposed to the enzyme xanthine oxidase and its substrate hypoxanthine. mtDNA repair was evaluated using quantitative Southern blot analysis. The results revealed that cells expressing ExoIII in mitochondria are deficient in mtDNA repair when compared with control cells that express ExoIII without MTS. This diminished mtDNA repair capacity rendered MDA-MB-231 cells more sensitive to oxidative damage, which resulted in a decrease in their long-term survival following oxidative stress. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Reactive oxygen species; Mitochondrial DNA repair; Oxidative stress; Exonuclease III; Breast cancer Abbreviations: IR, ionizing radiation; ROS, reactive oxygen species; mtDNA, mitochondrial DNA; ExoIII, Exonuclease III; APE, AP endonuclease; MTS, mitochondrial targeting signal; MOPS, 3-(N-morpholino)propanesulfonic acid; HEPES, N-2-hydroxyethyl- piperasine-N ′ -2-ethanesulfonic acid; XO, xanthine oxidase; HBSS, Hank’s balanced salt solution ∗ Corresponding author. Tel.: +1-251-460- 6765; fax: +1-251-460-8241. E-mail address: gwilson@usouthal.edu (G.L. Wilson). 1568-7864/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1568-7864(03)00019-3