Z Gastroenterol 2013; 51 - A18
DOI: 10.1055/s-0033-1347468

Different activating effect of genomic DNA from normal and tumorous epithelial cells on HT-29 carcinoma cells, HDF-Alpha fibroblasts and peripheral mononuclear blood cells

I Fűri 1, F Sipos 1, S Spisák 2, Á Patai 1, B Wichmann 2, G Műzes 1, O Galamb 2, G Valcz 1, A Kalmár 1, K Leiszter 1, K Tóth 1, B Barták 1, Z Nagy 1, B Molnár 1, Z Tulassay 1
  • 12nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary
  • 2Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary

Background: The methylation pattern of genomic DNA fragments may represent its organ origin. In case of tumorous and inflammatory conditions, the amount of cell-originated DNA is elevated in the serum. The mediation and immunobiologic effect of methylated DNA sequences on Toll-like receptor 9 expressing cells are not fully understood.

Aims: Our aims were to examine and compare the TLR9- and pro-inflammatory cytokine associated immunobiologic effect of human DNA from normal- and tumor origin on different cell types and ex vivo peripheral mononuclear cells (PBMCs). Methods: Genomic DNA was isolated from fresh frozen colonic epithelial samples. Subsequently, 5 × 105 HT-29 colon cancer cells, 2 × 105 HDF-alpha fibroblasts, and 2 × 106 PBMCs were incubated with 15 – 15 µg of normal or tumorous DNA for 6 hours. Total RNA was isolated by Qiagen RNeasy Mini kit. Expression levels of TLR9-signaling and pro-inflammatory cytokine related genes were measured by real time quantitative RT-PCR. Activation of PBMCs was determined by RT-PCR-based Qiagen T and B cell activation arrays.

Results: In TLR9 pathway, RT-PCR analysis of 13 genes showed altered mRNA expression on the level of key adaptor molecules. T and B cell activation array verified altered IL-2 mRNA expression. IL-2 was over-expressed after incubation with tumor-originated DNA. The treatment of HT29 cells with tumorous DNA resulted in higher expression of IL-1β than incubation with normal DNA. In case of HDF-alpha cells, normal DNA activated the whole TLR9-signaling pathway, in contrast to cells treated with tumorous DNA. In PMBCs treated with tumorous DNA higher IL-2 expression was observed compared it to PMBCs incubated with normal DNA

Conclusion: On different types of cells, exogenously administered DNA depending on its origin may display heterogenic pro-inflammatory signal. DNA from tumorous colonic epithelium could act as an endogenous pro-inflammatory ligand in HT29 cells. DNA from normal colonic epithelium may promote fibrogenesis of normal fibroblasts through TLR9 activation. The DNA-dependent activation of PMBCs may reflect to its role in mediating the observed immunobiologic effects.