Circulating cell‐free DNA release in vitro: kinetics, size profiling, and cancer‐related gene methylation

• Published in: Journal of cellular physiology 2019-08, Vol.234 (8), p.14079-14089
• Main Authors: Panagopoulou, Maria, Karaglani, Makrina, Balgkouranidou, Ioanna, Pantazi, Chrisoula, Kolios, George, Kakolyris, Stylianos, Chatzaki, Ekaterini
• Format: Article
• Language: English
• Subjects: Apoptosis; Azacitidine - pharmacology; Azacytidine; Biomarkers, Tumor - genetics; Biomaterials; Biomedical materials; Biopsy; Cancer; Cell culture; cell line; Cell-Free Nucleic Acids - drug effects; Cell-Free Nucleic Acids - genetics; cell‐free DNA; Deoxyribonucleic acid; DNA; DNA methylation; DNA Methylation - genetics; HeLa Cells; Humans; Kinetics; liquid biopsy; MCF-7 Cells; Methylation; Neoplasms - genetics; Neoplasms - pathology; Tumor cell lines
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.28097

Circulating cell‐free DNA (ccfDNA) is a biological entity of great interest due to its potential as liquid biopsy biomaterial carrying clinically valuable information. To better understand its nature, we studied ccfDNA in vitro in two human cancer cell lines MCF‐7 and HeLa. Normalized indexes of ccfDNA per cell population decreased over time of culture but were significantly elevated after exposure to IC50 doses of the demethylating/apoptotic agent 5‐azacytidine (5‐AZA‐CR). Fragment‐size profiling was indicative of active release, whereas exposure to 5‐AZA‐CR induced the release of additional shorter fragments, indicative of apoptosis. Finally, the methylation profile of a panel of cancer‐specific genes as assessed by quantitative methylation analysis in ccfDNA was identical to the corresponding genomic DNA and followed accurately changes caused by 5‐AZA‐CR. Overall, our in vitro findings support that ccfDNA can be a reliable biosource of clinically relevant information that can be further studied in these cell culture models. We studied circulating cell‐free DNA (ccfDNA) biology in vitro. Release decreased over time of culture, but was elevated after exposure to the demethylating/apoptotic agent 5‐azacytidine. Fragment‐size profiling was indicative of the active release and apoptosis. Finally, the methylation profile of cancer‐related genes reflected accurately the cell genomic DNA profile, supporting its use as a reliable biomarker