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.
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.
2nd Department of Internal Medicine, Semmelweis University, Szentkirályi Street 46, Budapest 1088, Hungary.
István
I
Fűri
2nd Department of Internal Medicine, Semmelweis University, Szentkirályi Street 46, Budapest 1088, Hungary.
Ferenc
F
Sipos
Molecular Medicine Research Unit, Hungarian Academy of Sciences, Nádor Street 7, Budapest 1051, Hungary.
Sándor
S
Spisák
1st Department of Pathology and Experimental Oncology, Semmelweis University, Üllői Street 26, Budapest 1085, Hungary.
Gergő
G
Kiszner
2nd Department of Internal Medicine, Semmelweis University, Szentkirályi Street 46, Budapest 1088, Hungary.
Barnabás
B
Wichmann
2nd Department of Internal Medicine, Semmelweis University, Szentkirályi Street 46, Budapest 1088, Hungary.
Andrea
A
Schöller
Molecular Medicine Research Unit, Hungarian Academy of Sciences, Nádor Street 7, Budapest 1051, Hungary.
Zsolt
Z
Tulassay
2nd Department of Internal Medicine, Semmelweis University, Szentkirályi Street 46, Budapest 1088, Hungary.
Györgyi
G
Műzes
Molecular Medicine Research Unit, Hungarian Academy of Sciences, Nádor Street 7, Budapest 1051, Hungary.
Béla
B
Molnár
D002454:Cell Differentiation / Q000502:physiology; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000096:biosynthesis / Q000235:genetics; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D007633:Keratins / Q000096:biosynthesis*; D053594:Myeloid Differentiation Factor 88 / Q000096:biosynthesis / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D048029:TNF Receptor-Associated Factor 6 / Q000096:biosynthesis / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*
C508606:CXCL8 protein, human; D016209:Interleukin-8; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; C495371:TLR9 protein, human; D048029:TNF Receptor-Associated Factor 6; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D051217:Toll-Like Receptor 9; D007633:Keratins; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; C507771:IRAK3 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases