30%. The percentage of LY1 cells selleck inhibitor in G1 phase increased from 33. 92% to 53. 74% after TSA treatment, while S phase cells declined from 49. 60% to 26. 60% after 24 h treat ment. However, in LY8 cells, the percentage of G2 phase cells increased from 17. 76% to 41. 65%, and S phase de creased from 45. 20% to 26. 80%, indicating a G2 M ar rest. A significant G0 G1 arrest was induced in DoHH2 cells after 24 h treatment relative to control cells, with a corresponding decrease of cells in S phase. A consistent induction of G0 G1 arrest and corresponding S phase reduction were observed in LY1 cells after 24 h treatment. However, we detected a G2 M arrest and relevant S phase decline in LY8 cells. The Annexin V PE 7AAD dual staining assay showed that 24 h treatment with TSA induced apoptosis in both LY1 cells and LY8 cells.

As shown in Figure 3B, significant apop tosis was induced in LY1 and LY8 cells after 24 h TSA exposure relative to control groups. Further more, apoptosis occurred earlier in LY8 cells than in LY1 cells. However, no significant apoptosis was observed in DoHH2 cells upon TSA treatment. HDAC expression in DLBCL cell lines We next determined the expression profile of the main HDAC isoforms in each cell line. Western blot analysis revealed differential expression levels of Class I HDACs and Class II HDACs in the three DLBCL lines. All three cell lines strongly expressed HDAC1 and HDAC2. Higher expression levels of HDAC3 and HDAC4 were found in DoHH2 and LY1 cells compared to LY8 cells. HDAC5 was only found in DoHH2 cells and at very high levels.

DoHH2 cells also expressed the highest levels of HDAC6, while moder ate to weak expression was observed in LY1 and LY8 cells. Together these data showed that the highest ex pression levels of all six HDAC isoforms were detected in DoHH2 cells, suggesting that the high sensitivity to TSA in DoHH2 cells might be due to the high expres sion of HDACs. TSA induced acetylation of histone and non histone proteins in DLBCL cells To further examine the effects of TSA, we evaluated acetylation of HDAC related biomarkers, histone H3 and tubulin. Histone H3 is one of the main substrates of Class I HDAC and tubulin is a target of HDAC6. Both acetyl histone H3 and acetyl tubulin levels were elevated in the three cell lines after 1 h treat ment, suggesting that TSA could inhibit their deacetylation.

Though a non histone protein, p53 is also a substrate of HDAC and its acetylation enhances its stability and extends its half life. Alterations of acetyl p53 levels were found in LY1 and LY8 cells. After 1 h incubation with TSA, acetyl p53 levels increased in LY1 and LY8 cells, which express mutant Cilengitide p53. In contrast, in DoHH2 cells, which express wild type p53, 50 nM TSA did not cause any apparent changes in acetyl p53 levels and downregulated p53 expression.