Pluripotent stem cells (PSCs) harbor exceptional potential for developing therapeutic advances for many diseases. However, when cultured in vitro, PSCs commonly acquire genetic abnormalities (e.g., aneuploidy). As these abnormalities can catalyze neoplastic progression, the genomic instability inherent to in vitro work has slowed efforts to bring PSCs from the bench to the bedside. Decatenation, the process of untangling chromosomes that become entwined during replication, contributes to the maintenance of genomic stability. Decatenation failure leads to a G2/M arrest via a distinct cell cycle checkpoint Activation of this checkpoint prevents aberrant daughter cell formation. It has been hypothesized that inefficient triggering of the decatenation checkpoint could be one cause of the observed genomic distortion. Recently, data has arisen demonstrating that the ataxia telangiectasia-mutated (ATM) kinase mediates this checkpoint, conflicting with older reports which posit that ATM has no part in executing cell cycle arrest in response to incomplete decatenation. ATM, a serine-threonine kinase, plays a major role in many intracellular signaling networks, such as the DNA damage response. Our lab has previously found that ATM, while present and active in PSCs, does not participate in canonical DNA damage repair pathways. Thus, we turned our attention to the decatenation checkpoint in order to elucidate ATM's role in PSCs. As we are in the process of developing induced pluripotent cells from normal and disease-specific fibroblasts, preliminary studies were conducted in human embryonic stem cells. Flow cytometry analysis showed that inhibition of decatenation activates ATM in PSCs and arrests them in G2. Inhibition of ATM using the highly-specific kinase inhibitor KU-60019 abrogates this arrest and allows PSCs to enter mitosis, resulting in bizarre chromosome morphology. Live cell imaging studies (accomplished preliminarily in non-transformed normal fibroblasts) revealed a prolonged metaphase-to-anaphase transition time after ATM inhibition. Combining inhibitors of decatenation and ATM resulted in failed karyokinesis and excessive anaphase bridge formation. After solidifying these findings, we will elucidate other proteins in the signaling cascade, specifically investigating if ATM interacts with Aurora-A and Plk1 while controlling this checkpoint. We will also determine if ATM inhibition for prolonged periods causes the development of aneuploidy using novel methods. In addition to its checkpoint role, there is evidence that ATM mediates apoptosis during certain stages of differentiation. As appropriate cell death is also important in preventing unwarranted growth, we aim to determine if ATM plays a role in apoptosis in PSCs. Western blotting and flow cytometry revealed that inhibition of ATM halts apoptosis at 3 and 6 hours, but increases it at 18 hours. Further investigation will clarify our preliminary studies. Studying specific protein interactions and mechanisms of genomic fidelity has led to the development of many chemotherapeutic agents and gives us insight into basic cellular operation. The goal of this proposal is to contribute to a greater understanding of processes that will impact future treatments and augment our understanding of cellular biochemistry.

Public Health Relevance

Inappropriate regulation of cell cycle checkpoints and apoptosis leads to malignant transformation. Additionally, effective therapies using pluripotent stem cells have been slow to develop due to genomic instability occurring when cultured ex vivo. This proposal aims to characterize the signaling involved in the maintenance of genomic stability in pluripotent stem cells. The results of this study could deepen our understanding of the catalysts of neoplastic transition, unveiling novel areas of modulation to prevent the development of cancer.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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Special Emphasis Panel (ZRG1-F05-R (20))
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Damico, Mark W
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Virginia Commonwealth University
Schools of Medicine
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