Histone deacetylase inhibitors on the other hand are presumed to

Histone deacetylase inhibitors on the other hand are presumed to function by increasing histone acetyla TSA induced apoptosis. These results indicate that external stimuli, that normally induce prolif erative or survival signalling pathways, cannot negate the apoptotic effect of TSA on CD4 T cells. TSA affects the NF reference 2 B signalling pathway The transcription factor NF B is a crucial regulator of immune and inflammatory responses, controlling the expression of a plethora of genes including cytokine, cytokine receptor, chemokine, cell adhesion molecule, and other cell surface receptor genes. Members of the NF B Rel family of transcription factors, such as NF B1, NF B2, RelA p65, RelB, and c Rel, are predominantly located in the cytoplasm of unstimulated cells.

Upon a variety of stimuli the inhibitory proteins known as I B proteins, which bind NF B proteins keeping them in a functionally inactive state, are specifically phosphorylated. This modification leads to ubiquitination and degradation of I B by the 26S proteosome and results in nuclear translocation of NF B and the activation of a variety of target genes. One such target is IL 2, suggesting that TSA might abrogate expression of this cytokine by inhibiting NF B mediated regulation. We investigated if decreased levels of nuclear NF B mediate the effect TSA has on IL 2 expression. West ern blot analysis of nuclear protein extracts of CD4 T cells exposed to growing concentrations of TSA shows a dose dependent drop in the level of nuclear NF B protein after stimulation with PMA ionomycin.

Thus, cells treated with 100 nM TSA had 3 4 fold lower levels of nuclear NF B protein as compared to control untreated cells, suggesting that TSA inhibits nuclear translocation of NF B. A recent report has shown that LPS induced translocation of NF B p65 from the cyto plasm to the nucleus in peripheral blood mononuclear cells is inhibited by butyrate. This effect is appar ently mediated by inhibition of I B degradation. Consequently, we investigated if the TSA mediated decrease in nuclear NF B was due to inhibition of I B degradation. Western blot analysis of cytoplasmic CD4 T cell protein extracts showed a drastic drop in I B levels just 15 min after stimulation with PMA ionomycin declining to even lower levels after 30 min, and maintain ing a low level of expression thereafter. This pattern was unaltered by TSA. We concluded that TSA Entinostat does not inhibit PMA ionomycin induced degra dation of I B, and that the decrease in the levels of nuclear NF B observed in TSA treated cells occurs by some mechanism other than inhibition of I B degradation.

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