In vivo analysis of circulating cell-free DNA release and degradation

Abstract

Cell-free DNA (cfDNA) was first detected in human plasma in the 1940s, but the knowledge on its regulation and rate of release is incomplete. CfDNA can originate from both normal and tumour cells. Our aims were to investigate the rate of cfDNA's release in SHO mice/HT-29 colorectal adenocarcinoma cell line xenograft model and to define the decay of methylated and non-methylated DNA fragments in C57BL/6 bloodstream. SHO mice were xenografted with human HT-29 cells, than blood samples were collected over 2 months. CfDNA was isolated, then quantified by real-time PCR with highly specific genomic and mitochondrial human and mouse primer sets. This method permitted to define the ratio of human/mouse DNA. To assess the degradation rate of cfDNA, 3000 bp sized methylated and non-methylated DNA fragments were injected into healthy and C38 tumour-cell vaccinated C57BL/6 mice's bloodstream. The decay of amplicons was measured with 19 PCR assays. The amount of human DNA until the 2nd week was below the limit of detection. From the third week, a continuous growth was experienced, which reached 18.26% by the 8th week. Moreover, it was found that in healthy animals the non-methylated DNA disappears from the plasma after 6 hours, while the methylated fragment was detectable even after 24 hours. In animals with tumour, both amplicons were detectable after 24 hours. The examination of the role and mechanism of cfDNA shows an increasing level of interest. This work can contribute to a better understanding of the release and degradation of cfDNA. Orv Hetil. 2018; 159(6): 223-233.