(B) HT-29 cells were pre-treated with 5-dAZA (50 μM) (black bars) or TSA (100 nM) (striped bars) for 24 hours (control) and then treated with LPS (50 ng/ml) and total RNA was extracted at the indicated time points after LPS administration and subjected to real time PCR analysis. The IL-8 mRNA levels were normalized to G6PD levels and expressed as relative to untreated control cells. Data points represent the average of triplicate determinations ± SD. Similar results were obtained in 3 independent experiments. Statistical analyses were performed compared to respective untreated control cells. *, p < 0.01; **, p < 0.05; absence of asterisks = not significant. Then we examined the effects of TSA and 5-dAZA on LPS Afatinib cell line induced

IL-8 expression (Figure 2B). HT-29 cells were primed with IFN-γ, pretreated for 24 hours with TSA or 5-dAZA, and then the cells were stimulated with 50 ng/ml LPS. We found that cells pretreated with 5-dAZA showed an IL-8 activation pattern very similar (p = n.s.) to that observed in cells treated with LPS alone Metformin research buy (Figure 1A), while TSA pretreatment significantly enhanced the LPS-mediated IL-8 activation (p < 0.05). Taken together

these data suggest that histone acetylation state but not DNA methylation may influence IL-8 expression in intestinal derived HT-29 cells. DNA methylation analysis of IL-8 promoter region Because the DNA methylation state at promoter regions may indeed influence the chromatin changes during gene activation, we sought to validate HT-29 cells as a good model to study chromatin modification at IL-8 locus. First, in order to confirm that DNA methylation is not involved in IL-8 gene regulation in HT-29 cells,

we analyzed the methylation state of 5 CpG sites lying around the IL-8 gene transcription start site (-83, -7, +73, +119, +191), both on upper and lower strands by MALDI-TOF analysis of genomic DNA extracted from untreated or LPS-treated cells (Figure 3A). We found that all five sites were completely unmethylated (0-2%) both in untreated and in LPS-treated cells (Figure 3B). Then, in order to investigate whether the observed DNA methylation profiles at the IL-8 locus were a specific feature of Cyclooxygenase (COX) HT-29 cell line or resembled those present in human tissues, we analyzed DNA from normal colon mucosa samples. Results showed that, similarly to HT-29 cells, CpG methylation at IL-8 locus in normal colon mucosa displayed an almost unmethylated state (0-4%) on both upper and lower strands (Figure 3B), confirming that HT-29 cells may be used to study chromatin dynamics at IL-8 gene. Interestingly, previous studies addressing the methylation state at IL-8 gene in several breast cancer cell lines, showed that the CpG sites located at the IL-8 promoter region were unmethylated in both metastatic and non-metastatic cell lines [20]. Figure 3 Methylation status of individual CpG sites on upper and lower strands at IL-8 promoter.