Association of Self-DNA Mediated TLR9-Related Gene, DNA Methyltransferase, and Cytokeratin Protein Expression Alterations in HT29-Cells to DNA Fragment Length and Methylation Status

Abstract

To understand the biologic role of self-DNA bound to Toll-like Receptor 9 (TLR9), we assayed its effect on gene and methyltransferase expressions and cell differentiation in HT29 cells. HT29 cells were incubated separately with type-1 (normally methylated/nonfragmented), type-2 (normally methylated/fragmented), type-3 (hypermethylated/nonfragmented), or type-4 (hypermethylated/fragmented) self-DNAs. Expression levels of TLR9-signaling and proinflammatory cytokine-related genes were assayed by qRT-PCR. Methyltransferase activity and cell differentiation were examined by using DNA methyltransferase (DNMT1, -3A, -3B) and cytokeratin (CK) antibodies. Treatment with type-1 DNA resulted in significant increase in TLR9 expression. Type-2 treatment resulted in the overexpression of TLR9-related signaling molecules (MYD88A, TRAF6) and the IL8 gene. In the case of type-3 treatment, significant overexpression of NFkB, IRAK2, and IL8 as well as downregulation of TRAF6 was detected. Using type-4 DNA, TRAF6 and MYD88A gene expression was upregulated, while MYD88B, IRAK2, IL8, and TNFSF10 were all underexpressed. CK expression was significantly higher only after type-1 DNA treatment. DNMT3A expression could also be induced by type-1 DNA treatment. DNA structure may play a significant role in activation of the TLR9-dependent and even independent proinflammatory pathways. There may be a molecular link between TLR9 signaling and DNMT3A. The mode of self-DNA treatment may influence HT29 cell differentiation.

Cell Free DNA of Tumor Origin Induces a ‘Metastatic’ Expression Profile in HT-29 Cancer Cell Line

“…Tumor DNA promoted CK20 and E-cadherin expression in the non-differentiated, HT—29 colon adenocarcinoma cells both on mRNA and protein level ( Table 4 , Fig 6C and 6F ) (p≤0.05). DNMT3a protein overexpression correlated with the previous results [ 28 ].…”

“…E-cadherin, DNMT3a based on our previous results. [ 28 ] PBS treated HT-29 cells showed weak membrane CK20 ( Fig 6A ), E-cadherin ( Fig 6D ) and weak/moderate cytoplasmic DNMT3a ( Fig 6G ) protein expression. Tumor DNA treatment induced increase in expression of CK20 strong positive (+++) pixels, ( Fig 6C ) E-cadherin weak (+) and moderate (++) positive pixels ( Fig 6F ) and DNMT3a weak (+) positive pixels ( Fig 6I ).…”

“…Based on our previous results, HT-29 human colon adenocarcinoma cells reflected altered DNA methylation level (via elevated DNMT3a methyltranferase activity) and CK20 epithelial marker expression after re—administration of self DNA. [ 28 ] In the present study we analyzed the autocrine and paracrine effects of DNA from tumor and healthy tissue on HT-29 cancer cells and fibroblasts by whole genomic mRNA expression analysis, and qRT PCR for validation of genes from TLR9 pathway. Furthermore immunocytochemical analysis was performed for selected differentiation markers, cell- adhesion molecules, and methyltransferases, for verification at the protein level following treatment with DNA from healthy and tumor tissues.…”

<p>DNA Methyltransferase Inhibitors: Catalysts For Antitumour Immune Responses</p>

“…The interaction of anticancer drugs with the host immune system has been implicated in therapeutic response 44. The major histocompatibility complex (MHC) class I is at the core of antigen presentation, and the expression of MHC class I molecules in tumour cells is often inhibited by irreversible mutations or reversible hypermethylation, resulting in downregulation 45. DNMTi can upregulate MHC class I levels in a variety of cancer tissues, as has been demonstrated in breast, lung, colon, and thyroid histotypes, as well as in human papilloma virus (HPV)-related cancers, sarcomas, and gliomas,46–50 and they promote the release of interferon-γ from tumour-specific cytotoxic T lymphocytes (CTLs), which kill target cells 51.…”

Extracellular Self-DNA (esDNA), but Not Heterologous Plant or Insect DNA (etDNA), Induces Plasma Membrane Depolarization and Calcium Signaling in Lima Bean (Phaseolus lunatus) and Maize (Zea mays)

“…In animal cells, different DNA pattern recognition receptors and sensors have been identified. These include among others cyclic GMP–AMP (cGAMP) synthase (cGAS) [ 15 ], DNA-sensing inflammasome receptor absent in melanoma 2 (AIM2) [ 16 ], cationic antimicrobial peptide LL37 [ 17 ], and self-DNA bound to Toll-like receptor 9 (TLR9) [ 18 , 19 , 20 ].…”