Publication Date

2025

Document Type

Thesis

Committee Members

Michael George Kemp, Ph.D. (Advisor); Ravi P. Sahu, Ph.D. (Committee Member); Yong-Jie Xu, M.D., Ph.D. (Committee Member)

Degree Name

Master of Science (MS)

Abstract

Cell-free DNA (cfDNA) can arise from dying cells or be actively secreted by living cells, and is found either unbound, within extracellular vesicles, or in other macromolecular complexes. cfDNA plays roles in intercellular communication and immune responses and is clinically used for diagnostics through PCR and DNA sequencing. Interestingly, our lab has detected lesions in cfDNA after treatment with DNA-damaging agents including UVB and cisplatin. These findings prompted further investigation into which DNA lesions and labels can be found in the cell-free space. Importantly, we sought a mechanism of labeling DNA with a synthetic nucleoside to enable robust tracing using fluorescent labels and sensitive detection of low-abundance cfDNA. In this study, we evaluated the nucleoside analogs BrdU (bromodeoxyuridine), EdU (5-ethynyl- 2'-deoxyuridine), and F-ara-EdU (2-deoxy-2-fluoro-5-ethynyluridine) for labeling cfDNA. Among these, EdU generated strong signals even without UVB treatment but reduced cell viability and induced caspase-dependent apoptosis, highlighting its cytotoxic effects. Notably, blocking apoptosis with the pan-caspase inhibitor zVAD-FMK did not reduce EdU-labeled cfDNA release, suggesting that cfDNA can be released through alternative, caspase-independent pathways, such as active secretion. In contrast, F-ara-EdU produced robust signals after UVB exposure without affecting cell viability, making it a safer and more reliable option for studies where minimizing toxicity is important. In summary, these data demonstrate the advantages and disadvantages of different nucleoside analogs for tracking cfDNA.

Page Count

63

Department or Program

Department of Pharmacology and Toxicology

Year Degree Awarded

2025

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