The outer layeror cap layerof the TEB contains a special population of pluripotent stem cellswhich in the end identify
The ability to do so was illustrated using a highly aggressive CMS4 subline, which became more responsive to IRF-8 induction following exposure to TSA or DP alone or in combination with IFN-c. Although it remains to be fully investigated why the two cell lines varied in their response to IRF-8 induction, these data nonetheless provide evidence that IRF-8 is a key component for response to HDACi. Future studies will also determine whether the epigenetic profile of the IRF-8 promoter is different in CMS4 vs. CMS4-met.sel cells, which may help to explain in part their differential responsiveness of IRF-8 induction to TSA treatment. To further demonstrate the importance of IRF-8 in this model, we examined the effects of TSA on IRF-8 promoter activity using a reporter assay. It is important to note that this IRF-8 promoter construct contains the endogenous DNA sequence without any hypermethylation or HDAC sites. Thus, these experiments were designed not only to substantiate the effect of TSA on IRF-8 expression, but also to determine whether the effect of TSA on IRF-8 promoter activity was HDAC-dependent. We hypothesized that if the acetylation status of IRF-8 matters for response to TSA, then an IRF-8 promoter sequence lacking HDAC sites would be unresponsive to TSA treatment. We found that TSA alone and more so in combination with IFN-c increased IRF-8 promoter activity in both parental and aggressive CMS4 cells. For both cell lines after TSA treatment, the IRF-8 patterns seen at the promoter level paralleled the IRF-8 patterns observed at the mRNA level. It is interesting to note, however, that since the exogenous promoter fragment did not contain deacetylation sites, these data suggest that TSA could modulate IRF-8 transcription via mechanisms not necessarily related to HDAC inhibition at the promoter level. We next examined the integrity of events upstream of IRF-8, mainly STAT1 as it is known to be essential for IFN-c-inducible gene regulation, including IRF-8. Phosphorylation of STAT1 plays an important role in regulating IFN-c-mediated gene induction. It has also been reported that HDACi, such as TSA, alters the expression of IFN-c-inducible genes through acetylation of STAT1 in myeloid cells and tumor cells. We found that STAT1 silencing in either parental or aggressive CMS4 cells led to a significant reduction in TSA- or IFN-cinduced IRF-8 promoter activity, the latter of which served as a positive control. These results suggested that TSA-induced IRF-8 promoter activity was STAT1-dependent. However, it is important to emphasize that single agent TSA treatment did not seem to elicit STAT1 phosphorylation, but did promote STAT1 acetylation. Thus, we posit that TSA may impact STAT1 function in an unphosphorylated manner, as previously reported in other systems. Overall, our data are consistent with a model that tumor-cell expression of IRF-8 is integral for HDACi-induced antitumor activities. HDACi exposure may render neoplastic cells more receptive to IRF-8 induction and Fas-mediated death under pro-inflammatory conditions. Therefore, IRF- 8 transcription may be influenced in two ways; one by IFN-c and the other by HDACi. In either case, IRF-8 transcription is STAT1-dependent. STAT1 activation, however, may result from both phosphorylation-dependent and -independent mechanisms, which warrant further study. Moreover, these data do not preclude the possibility that the IRF-8 promoter may be regulated by multiple epigenetic mechanisms, including DNA methylation, and that these mechanisms may impact IRF-8 expression and consequently Fas sensitivity in a direct or indirect manner. Such complex issues, therefore, warrant further study. Nonetheless, the induction of IRF-8, in turn, modulates tumor response to immune attack via Fas-mediated apoptosis. Based on observations in myeloid leukemia, IRF-8 may regulate Fas responsiveness by acting as a transcriptional activator of pro-apoptotic genes, such as caspases, and/or a transcriptional repressor of anti-apoptotic genes, such as PTPN13 or members of the Bcl-2 family. Altogether, our results point to IRF-8 expression in tumors as being a potential biomarker for efficacy of response to HDACi and a possible molecular target to improve response to therapy. Plants produce a wide range of secondary metabolites, or allelochemicals, to protect themselves from devastation by animals, insects and pathogens. Furanocoumarins are one group of naturally occurring plant secondary compounds that have shown toxicity against a broad spectrum of animals including herbivorous insects, and affect their feeding behavior.