The Effect of Protein Transport Inhibitors on the Production of Extracellular DNA Evgeniy S. Morozkin, Tatyana I. Babochkina, Valentin V. Vlassov, and Pavel P. Laktionov Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia The presence of various genomic sequences in the pool of extracellular DNA was studied by cloning and sequencing the DNA eluted from the surface of HeLa cells. Sequences of 19 genes, 10 pseudogenes, and 41 repeated elements were found in 103 clones. Sequences of LINE repeats were found in 17% of the clones; consequently real-time PCR assay for quantification of LINE repeats was chosen to study the influence of protein transport inhibitors on the concentration of free and cell-surface-bound DNA in the cultures of HUVEC and HeLa cells. Treatment of both cell lines with the inhibitors did not in- terfere with the concentration of extracellular DNA in the growth medium, except for chloroquine, which doubled the concentration of extracellular DNA in HUVEC culture. The treatment of HUVECs with monensin, glyburide, or methylamine decreases the cell-surface-bound DNA concentration by 30, 35, and 19%, respectively. The incubation of HeLa cells with monensin reduces the concentration of cell-surface-bound DNA by 15%; however, the treatment with glyburide increases cell-surface-bound DNA concen- tration by 50%. The data obtained demonstrate the involvement of vesicular transport in generation of extracellular DNA. Key words: extracellular DNA; DNA sequencing; inhibition of protein transport Introduction The mechanisms of DNA release into extra- cellular media are poorly characterized. The electrophoretic pattern of circulating DNA is similar to that in apoptotic/necrotic cell cul- tures; thus, apoptosis and necrosis are regarded as the main sources of extracellular DNA (exDNA). 1,2 There are data demonstrating that living cells excrete DNA. Along with some bac- teria, which produce substantial quantities of exDNA, 3 eukaryotic cells have also been shown to excrete DNA during their normal growth: in particular, DNA is excreted by human lym- phocytes after mitogenic stimulation 4 and by somatic cells of copepods during chromatin diminution. 5 Address for correspondence: Evgeniy S. Morozkin, Institute of Che- mical Biology and Fundamental Medicine, Siberian Division of the Rus- sian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk, 630090 Russia. Voice: +7-383-3304654; fax: +7-383-3333677. morozkin@niboch.nsc.ru The contribution of active DNA secretion by living cells to the production of exDNA can be evaluated using inhibitors of cell secretory mechanisms. In this work, we have studied the influence of inhibitors of classical and nonclas- sical protein secretion pathways on the con- centration of exDNA in the cultures of human umbilical vein endothelial cells (HUVECs) and human cervical carcinoma (HeLa) cells. Materials and Methods Cultivation of Cells and Extracellular DNA Isolation HUVECs were obtained by treatment of human umbilical cord veins with 0.1% col- lagenase. 6 HUVECs and HeLa cells were cultivated in DMEM culture medium sup- plemented with 10% FBS, 100 μg/mL streptomycin, and 100 μg/mL penicillin at Ann. N.Y. Acad. Sci. 1137: 31–35 (2008). C  2008 New York Academy of Sciences. doi: 10.1196/annals.1448.026 31