Indiana University-Purdue University Indianapolis (IUPUI) / DNA double strand breaks (DSB), particularly those induced by ionizing radiation (IR) are complex lesions and if not repaired, these breaks can lead to genomic instability, chromosomal abnormalities and cell death. IR-induced DSB often have DNA termini modifications including thymine glycols, ring fragmentation, 3' phosphoglycolates, 5' hydroxyl groups and abasic sites. Non-homologous end joining (NHEJ) is a major pathway responsible for the repair of these complex breaks. Proteins involved in NHEJ include the Ku 70/80 heterodimer, DNA-PKcs, processing proteins including Artemis and DNA polymerases µ and λ, XRCC4, DNA ligase IV and XLF. The precise molecular mechanism of DNA-PK activation and Artemis processing at the site of a DNA DSB has yet to be elucidated. We have investigated the effect of DNA sequence and structure on DNA-PK activation and results suggest a model where the 3' strand of a DNA terminus is responsible for annealing and the 5' strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in microhomology-mediated NHEJ. Artemis, a nuclease implicated in processing of DNA termini at a DSB during NHEJ, has been demonstrated to have both DNA-PK independent 5'-3' exonuclease activities and DNA-PK dependent endonuclease activity. Evidence suggests that either the enzyme contains two different active sites for each of these distinct processing activities, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. These results reveal novel mechanisms of two critical NHEJ proteins, and further enhance our understanding of DNA-PK and Artemis activity and their role in NHEJ.

The type 1 insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase that mediates diverse cellular functions including growth, differentiation, migration and apoptosis protection. IGF-1R signalling has been implicated in tumorigenesis in a variety of cancers, and IGF-1R inhibitory drugs are currently undergoing clinical evaluation. Previous work in our laboratory has shown IGF-1R over-expression in urological cancers at both the mRNA and protein level, thus making it a potential therapeutic target. The first aim of this project was to develop a protocol for IGF-1R immunohistochemistry, investigate the expression and cellular distribution of the IGF-1R receptor in clear cell renal cell carcinomas (ccRCC), and assess correlation with clinical parameters. In tissue microarray analysis, IGF-1R was detected in ~90% of 195 ccRCCs, with signal in the plasma membrane, cytoplasm and also in the nucleus. The presence of nuclear IGF-1R in up to 50% of ccRCCs and its association with adverse prognosis was a novel finding, and suggests that nuclear IGF-1R may influence ccRCC biology. Further investigations will clarify its role in the nucleus and its potential as a prognostic biomarker. The second aim was to investigate effects of IGF-1R inhibition on radiosensitivity and DNA repair, following previous work in our laboratory showing that IGF-1R depletion enhances chemo- and radio-sensitivity, delays double strand break (DSB) resolution, and may play a role in the homologous recombination (HR) pathway of DNA DSB repair. However, the repair defect seen in these early experiments was larger than could be entirely explained by a defect in HR. The current project used a small molecule IGF-1R tyrosine kinase inhibitor AZ12253801 (AstraZeneca), which blocked IGF-1 induced IGF-1R activation and inhibited cell survival. AZ12253801 enhanced the radiosensitivity of prostate cancer cells, which appeared to be independent of effects of IGF-1R inhibition on cell cycle distribution and apoptosis induction. IGF-1R inhibition delayed the resolution of γH2AX foci, supporting a potential role for the IGF-1R in DSB repair. This delay in focus resolution was apparent at early time-points (less than 4 hr), and was epistatic with DNA dependent protein kinase (DNAPK) inhibition in prostate cancer cells and DNAPK deficiency in glioblastoma cells. These results suggest a role for the IGF-1R in the non-homologous end-joining (NHEJ) pathway of DNA DSB repair. A cell-based reporter assay in HEK-293 cells confirmed that IGF-1R inhibition suppressed DSB repair by NHEJ, helping to explain the radiosensitization demonstrated upon IGF-1R inhibition. There was lack of support for a transcriptional effect, with no significant change observed in gene expression on microarray analysis. Although the mechanism of this effect remains unclear, the observed inhibition of NHEJ has implications for the use of IGF-1R inhibitors in combination with DNA damaging agents in cancer treatment.