Role of polynucleotide kinase/phosphatase in mitochondrial DNA repair

Mutations in mitochondrial DNA (mtDNA) are implicated in a broad range of human diseases and in aging. Compared to nuclear DNA, mtDNA is more highly exposed to oxidative damage due to its proximity to the respiratory chain and the lack of protection afforded by chromatin-associated proteins. While repair of oxidative damage to the bases in mtDNA through the base excision repair pathway has been well studied, the repair of oxidatively induced strand breaks in mtDNA has been less thoroughly examined. Polynucleotide kinase/phosphatase (PNKP) processes strand-break termini to render them chemically compatible for the subsequent action of DNA polymerases and ligases. Here, we demonstrate that functionally active full-length PNKP is present in mitochondria as well as nuclei. Downregulation of PNKP results in an accumulation of strand breaks in mtDNA of hydrogen peroxide-treated cells. Full restoration of repair of the H2O2-induced strand breaks in mitochondria requires both the kinase and phosphatase activities of PNKP. We also demonstrate that PNKP contains a mitochondrial-targeting signal close to the C-terminus of the protein. We further show that PNKP associates with the mitochondrial protein mitofilin. Interaction with mitofilin may serve to translocate PNKP into mitochondria

Synthetic Lethal Targeting of PTEN-Deficient Cancer Cells Using Selective Disruption of Polynucleotide Kinase/Phosphatase

A recent screen of 6,961 siRNAs to discover possible synthetic lethal partners of the DNA repair protein polynucleotide kinase/phosphatase (PNKP) led to the identification of the potent tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Here, we have confirmed the PNKP/PTEN synthetic lethal partnership in a variety of different cell lines including the PC3 prostate cancer cell line, which is naturally deficient in PTEN. We provide evidence that codepletion of PTEN and PNKP induces apoptosis. In HCT116 colon cancer cells, the loss of PTEN is accompanied by an increased background level of DNA double-strand breaks, which accumulate in the presence of an inhibitor of PNKP DNA 3'-phosphatase activity. Complementation of PC3 cells with several well-characterized mutated PTEN cDNAs indicated that the critical function of PTEN required to prevent toxicity induced by an inhibitor of PNKP is most likely associated with its cytoplasmic lipid phosphatase activity. Finally, we show that modest inhibition of PNKP in a PTEN knockout background enhances cellular radiosensitivity, suggesting that such a "synthetic sickness" approach involving the combination of PNKP inhibition with radiotherapy may be applicable to PTEN-deficient tumors

Photo-activation of the delocalized lipophilic cation D112 potentiates cancer selective ROS production and apoptosis.

Delocalized lipophilic cations (DLCs) selectively accumulate in cancer cell mitochondria and have long been explored for therapeutic applications. Although targeted effects to cancer cells are demonstrated in vitro, non-specific toxicities in vivo have hampered clinical development. Identifying the molecular mechanisms of action and enhancing selectivity are thus necessary next steps to improve these compounds and evaluate their suitability for further drug development. D112 is one such DLC with promising properties. We previously demonstrated that D112 selectively induced intrinsic apoptosis in transformed versus non-transformed cell lines. Here we show that D112 preferentially entered transformed cells where it interacted with, and damaged mitochondrial DNA, inhibited Complex I respiration and induced reactive oxygen species (ROS). ROS production was critical for Bax activation and subsequent apoptosis. Importantly, photo-activation of D112 potentiated selective ROS production and increased the window of toxicity towards cancer cells over non-transformed cells. Thus photodynamic therapy would be an exciting adjunct to D112 studies and may be generally applicable for other DLCs that are currently under therapeutic investigation