DISSERTATION CHARACTERIZATIONS OF CHROMOSOME ABERRATIONS, TELOMERE DYSFUNCTION, AND RADIOSENSITIVITY SIGNATURES IN CANINE CANCER CELL LINES Submitted by Junko Maeda Graduate Degree Program in Cell and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2015 Doctoral Committee: Advisor: Takamitsu Kato Joel Bedford Susan Bailey Douglas Thamm Copyright by Junko Maeda 2015 All Rights Reserved ABSTRACT CHARACTERIZATIONS OF CHROMOSOME ABERRATIONS, TELOMERE DYSFUNCTION, AND RADIOSENSITIVITY SIGNATURES IN CANINE CANCER CELL LINES Cancer is now a leading cause of death in dogs as well as in humans due to longer life spans resulting from advances in nutrition and veterinary medicine, and the higher expectations of pet owners. Companion dogs share environmental influences with humans and spontaneously develop tumors with histopathologic and biologic behavior similar to tumors that occur in humans. Therefore, canine cancer research has much potential to benefit both dogs and humans. Cancer cell lines have been widely used as in vitro experimental model systems and have proven to be useful for exploring the underlying biology of cancer. Canine cancer cell lines have increasingly been developed and utilized, but are not as fully characterized as human cell lines. In this thesis, we characterized canine cancer cell lines by examining: 1) chromosome aberrations, long appreciated as valuable biomarkers of carcinogenesis; 2) telomeres, chromosomal features with important implications for both aging and cancer; and 3) cellular radiosensitivity signatures, critical for evaluating individual response to radiation therapy. Such characterization of canine cancer cell lines will provide a better understanding of underlying canine cancer biology and provide new insights into improved clinical management, such as development of novel therapeutic targets and identification of radiation sensitivity markers, not only for dogs, but of potential relevance for humans as well. ii First, we investigated chromosome and telomere aberrations in canine osteosarcoma (OSA) cell lines, a common primary bone tumor in both humans and dogs. Previously, malignant canine cancer cells have been reported to exhibit metacentric chromosomes (morphologically irregular chromosomes for canines) in a range of canine tumors including osteosarcoma. The metacentric chromosomes, which likely represent end-to-end chromosomal fusion events (Robertsonian translocations), may reflect telomere dysfunction, leading us to hypothesize that chromosome aberrations involving uncapped telomeres may be a common feature of canine OSA cells. Eight established canine OSA cell lines were evaluated; chromosome number and frequency of metacentric chromosomes were determined in metaphase spreads. Using fluorescence in situ hybridization (FISH) with a telomere specific probe, the contribution of dysfunctional telomeres was characterized. We also assessed telomere associated factors including telomerase activity, co- localization of DNA damage and telomere signals (telomere-dysfunction induced foci, TIFs), and expression of a DNA repair protein shown to be required for mammalian telomeric end-capping function, specifically the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Despite variable chromosome numbers, proliferation rates and radiosensitivities, all eight canine OSA cell lines displayed increased numbers of metacentric chromosomes and exhibited numerous telomere aberrations including interstitial telomeric signals and telomere fusions (telomere signals at the point of fusion, in this case in the centromeric regions). D17, the oldest canine OSA cell line used in the study, showed the highest frequency of telomere aberrations (51.4 per cell) and metacentric chromosomes (43.2 per cell). Furthermore, the cell lines were all telomerase positive and showed no correlations between their telomere aberrations and the other telomere associated factors analyzed. To better characterize the telomere dysfunction associated with canine OSA, we investigated telomere aberrations in primary cultures from ten spontaneous canine OSAs, as well iii as the effects of long-term culture. Seven of the primary samples displayed no increase in frequency of metacentric chromosomes, while three of the samples did have elevated levels (11.5 per cell in the highest primary culture); however, no telomere signals were present at the involved centromeric regions. Telomere aberrations were observed in all of the primary cultures, but the number was small (1.87 per cell in the highest primary culture). Interestingly, we found that metacentric chromosome frequencies increased in one primary OSA culture with increasing passage in culture. In contrast, metacentric chromosomes did not accumulate with long-term culture of non-cancerous canine fibroblasts, or DNA repair deficient mouse fibroblasts (homozygous ataxia telangiectasia mutated (ATM) gene deficiency). Together, these results suggested that metacentric chromosomes and telomere dysfunction of are characteristics of canine OSA. Therefore, targeting of these unique telomere aberrations has potential for both improving diagnosis of canine OSA, as well as development of novel treatment strategies. Lastly, we investigated radiosensitivity in a panel of canine cancer cell lines representing different cancer types. A human cancer cell line panel, NCI-60, consisting of different tumor types has been successfully utilized for in vitro anticancer drug screening, demonstrating the utility of such an approach in understanding cancer mechanisms; gene expression profiling and variable radiosensitivities facilitated development of informative markers regardless of tumor tissue type. A canine cancer cell line panel, called the ACC30, was recently developed at the Flint Animal Cancer Center (FACC), Colorado State University and includes microarray gene expression data. We aimed to test the hypothesis that identification of the determinants of response to ionizing radiation in the diverse canine cancer cell lines would provide additional information, some possibly specific to dogs, and some potentially supplementing those reported for human cancer. We obtained 27 canine cell lines derived from ten tumor types from the ACC30 panel. First, iv radiosensitivity was determined using a clonogenic assay for adherent cell cultures, or a limiting dilution assay for suspension cell cultures. The 27 cell lines had varying radiosensitivities regardless tumor type (survival fraction at 2 Gy, SF2= 0.2–0.9). Based on the cellular characteristics analyzed in this study, there was a moderate correlation between radioresistance and better plating efficiency in the 27 cell lines. Next, we selected the six most radiosensitive cell lines as the radiosensitive group and the five most radioresistant cell lines as the radioresistant group. Then, we evaluated known parameters for cell killing by ionizing radiation (IR) including IR-induced DNA double strand break (DSB) repair and apoptosis, in the radiosensitive group as compared to the radioresistant group. However, the two groups were not distinguished by these parameters. Further, we investigated a possible common radiosensitivity signature using the basal gene expression profiling of the ACC panel for 20,000 genes. More than 550 genes were identified as being differentially expressed between the radiosensitive and radioresistant groups. Gene set enrichment analysis was used to inform potential pathways and functions involving the differentially expressed genes and indeed, several biological processes including cell adhesion, cell migration and apoptosis were related to radiosensitivity in the canine cancer cell lines. In support of our findings, cell adhesion was one of the signatures previously identified in the human microarray analysis. Together, our results suggest that cell adhesion related genes, rather than the more commonly regarded radiosensitivity associated apoptosis and DNA repair related genes, may provide beneficial radiosensitivity biomarkers for predicting individual response to radiotherapy, regardless of tumor type. Thus, the radiosensitivity signatures characterized here may help guide future development of intrinsic tumor radiosensitivity biomarkers for predictive assays in canine cancer, and has the potential to improve predicting radiosensitivity in human cancer as well. v ACKNOWLEDGEMENTS I would like to start by first thanking the members of my graduate committee: Dr. Takamitsu Kato, Dr. Susan Bailey, Dr. Joel Bedford, and Dr. Douglas Thamm for their help and guidance throughout the project. I would especially like to thank my advisor Dr. Takamitsu Kato for his support, encouragement, and guidance over the last four years, without whom, none of the work would have been possible. I would also like to thank all the lab members in the Kato lab. In particular, I would like to thank Colleen Brents, Coral Froning, Erica Roybal, Garret Phoonswadi and Garret Rota for the effort, time and energy they have committed to the completion of this project. I would like to thank Dr. Ian Cartwright for his previous support in Dr. Kato’s lab. A special thanks to Dr. Hatsumi Nagasawa and Barbara Rose for their help on the cell culture experiments. Lastly, I would like to thank the faculty and staff of the Department of Cell and Molecular Biology, Environmental Radiological Health Sciences and Flint Animal Cancer Center. Especially I would like