Methylated Cell-Free DNA In Vitro and In Vivo

We have investigated the stability, circulation and generation of methylated DNA (methDNA) in vivo and in vitro. In serum, the methDNA (free and as nucleoprotein complexes circulating in human blood) was shown to degraded more slowly then unmethylated DNA (unmethDNA). Residual free methDNA circulates in blood for longer than unmethDNA although it is eliminated faster from blood immediately after injection. After injection of human plasma in mice via the caudal vein, human circulating DNA was shown to circulate in blood for a long time, to accumulate in tissues and to diffuse back into the blood. A comparative, quantitative study of RARbeta2 gene methylation in cell-free DNA (cfDNA) and genomic DNA (gDNA) of primary and transformed cells was made using methylation-specific quantitative PCR (MS-qPCR) and pyrosequencing. The MS-qPCR data demonstrate overrepresentation of methDNA sequences in cfDNA of primary cells. Methylation level (ML) of most CpG-sites in cfDNA and gDNA is similar, but certain sites are overmethylated, the extent of their methylation differing between cfDNA and gDNA. Thus, a detailed investigation of methylated cfDNA generation and factors influencing its stability, circulation and elimination from the blood are required for a rational selection of aberrantly methylated tumor markers.