159P - Effects of Modified Self-Dna Sequences on Cell Kinetic Parameters and Differentiation of Ht29 Cancer Cells

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ABSTRACT Aim: We have recently reported that Toll-like receptor 9 (TLR9)-signaling pathway activiation by modified self-DNA depends on the structural characteristics of DNA sequences. However, no concrete data exists on how the characteristics of TLR9 activating self-DNA fragments influence the viability, proliferation, death, and differentiation of cancer cells, which all may have important immunobiologic consequences in inflammatory and tumorous colonic disorders. To understand the biologic role of modified self-DNA bound to TLR9, we assayed its effect on cell viability, proliferation, death and differentiation in HT29 colon cancer cells. Methods: Self-DNA from HT29 cells was extracted and artificially fragmented (with ultrasonic fragmentation) and/or hypermethylated (using CpG methyltransferase M.SssI). HT29 cells were then incubated separately with type-1 (normally methylated/non-fragmented), type-2 (normally methylated/fragmented), type-3 (hypermethylated/non-fragmented), or type-4 (hypermethylated/fragmented) self-DNAs for 24, 48 and 72 hours. Cell viability was examined by MTT-assay, cell proliferation by TUNEL method, cell proliferation by Ki-67 assay, while cell differentiation by pancytokeratin (CK) immunohistochemistry. Results: Treatments with type-1, -3 and -4 DNA sequences resulted in remarkable decrease of cell viability and proliferation, and increase of apoptosis after 72h. The effects of type-3 DNA incubation were the most significant. Type-2 DNA treatment resulted in a slight decrease of cell viability after 48h, but at 72h all parameter of cell kinetics were similar to that of control cells. Regarding cell differentiation, CK expression was only increased after type-3 DNA treatment. Conclusions: Activation of TLR9-signaling pathways by modified self-DNA sequences may display significant effects on cell kinetic parameters like viability, proliferation or death, moreover may influence cell differentiation, which may have clinical importance in the future. Disclosure: All authors have declared no conflicts of interest.