Radiosensitization of human cancer cell lines in vitro by genetic targeting of Ku70 and the PI3 -kinase/Akt pathway

In the transformation to a cancerous phenotype, cells often develop resistance to chemotherapeutic drugs and ionizing radiation used for cancer therapy. The P13-K/Akt pathway is a pro-growth, anti-apoptotic pathway downstream of receptor tyrosine kinases that is frequently dysregulated in tumor cells. Our hypothesis was that inhibition of PI3-K/Akt by adenoviral-delivery of the tumor suppressor PTEN would radiosensitize human tumor cell lines in vitro. Three cell lines were radiosensitized by our Ad-PTEN treatment as determined by clonogenic survival. DNA double strand break (DSB) repair was tracked in one of the sensitized cell lines by counting the number of γ-H2AX foci microscopically. The Ad-PTEN treated cells were not as efficient at DNA repair as the controls. We believe this repair inhibition results in a mitotic catastrophe upon attempted cell division, and accounts for the radiosensitive phenotype we observed. The second project targeted the protein Ku70, an early component of the DNA DSB repair process that tethers damaged DNA ends, and activates DNA-PK. We hypothesized that reduction of Ku70 protein levels using siRNA would lead to radio sensitization caused by inhibition of the repair of DNA DSBs. We observed a delay in the repair kinetics of cells with partially depleted Ku70, and conclude that the cells entered mitosis prematurely, with unrepaired DSBs, and either died or senesced. In addition to having a role in DSB repair, the DNA-PK catalytic subunit performs an essential phosphorylation on Akt Ser-473. Due to the Ku70/80 subunit\u27s role in activating DNA-PK, we hypothesized that Ku70 inhibition would result in a decreased phosphorylation of Akt Ser-473. We observed that levels of phospho-Akt Ser-473 were decreased when we depleted Ku70 with siRNA. These data fit a model where Ku70 is bound to DNA-PKcs and acts as an activator or scaffold protein for DNA-PKcs. We conclude that depletion of Ku70 levels inhibits Akt activity, and inhibition of Ku70 and PI3-K/Akt signaling may radiosensitize cells by inhibiting DNA DSB repair