Due to the ple otropic effects on gene expression induced by HDACi, we first confirmed that our selected qPCR normalizing gene was not modulated by HDACi treatment in either cell line prior to DEG verification. We selected 2 house sellekchem keeping genes, 18s rRNA and GAPDH, whose expression was unchanged in the microarray analysis and confirmed using qPCR that these genes retained consist ent expression during HDACi treatment. GAPDH was subsequently used to normalize all qPCR data. To further validate and characterize the DEGs iden tified by the microarray analysis, we analyzed the time dependent change in expression of the selected DEGs at 6, 12 and 24 hours post HDACi treatment when compared to vehicle treated time matched controls.

The pattern of expression obtained for 14 of the 15 selected DEGs 24 h post treatment showed consistent directional conforma tion and cell line specific modu lation between the qPCR and microarray analyses. THBS 1, AVEN and AURKB demonstrated significant cell line specific changes in expression at 24 h as observed in the microarray analyses. HIST1H1C was ini tially identified as consistently upregulated by the micro array analysis, but subsequent qPCR analysis indicated this gene to be consistently downregulated with either HDACi in both cell lines. QPCR validation of our core panel of 11 genes also demonstrated consistent modulation at 24 h with the microarray analysis. In addi tion, these 11 genes also demonstrated time dependent changes in expression at 6 and/or 12 h post HDACi treat ment.

However, in several instances, the fold changes obtained by qPCR were significantly higher for several genes than those obtained in the microarray analyses, particularly for the more heavily regulated genes as previously reported. For example, DHRS2 was induced by 5 fold in both cell lines following HDACi treatment in the microarray GSK-3 analysis. Subsequent qPCR analysis determined the fold increase in DHRS2 tran scripts to be in the order of 36 97 fold in HT29 cells and 226 445 fold in the HCT116 cells. Similarly, thymidylate synthase was down regulated by HDACi in both cell lines 2. 7 3. 8 fold in the microarray analysis, whereas qPCR determined that HDACi treat ment induced a 30 fold downregulation of TYMS 24 h post treatment in both cell lines.

Discussion In an effort to characterize the response of colon cancer cells to HDACi, we analyzed the gene expression profile of two colon cancer cell lines following treatment with two HDACi, vorinostat and LBH589. Both HDACi resulted in significant inhibition of tumor cell proliferation, an accu mulation of acetylated histones and the onset of apoptotic cell death. However, LBH589 exerted done antiproliferative effects at significantly lower concentrations than vorinos tat, consistent with previous reports utilizing these HDACi. Specifically, the IC50 for LBH589 was in the single digit nanomolar range while vorinostat required concentrations in excess of 1 M.