CG-1521 also induced the expression of genes associated with the negative regulation of apoptosis (GO:0006629), response to unfolded protein (GO:0016491) and response to stress (GO:0009055) (Table 2 and Fig.?5B). of the genes are commonly modulated in both cell lines, suggesting that CG-1521 and TSA target different biological processes in the two cell lines most likely though the inhibition of different HDACs in these cell lines. Gene ontology (GO) analysis reveals that CG-1521 affects the expression of mRNAs that encode proteins associated with the spindle assembly checkpoint, chromosome segregation, and microtubule-based processes in both cell lines and has cell-type specific effects on lipid biosynthesis, response to DNA damage, and cell death. < 0.05 (*). NS, not significant. Effect of CG-1521 and TSA on cell cycle kinetics and apoptosis in IBC cells To investigate the underlying mechanism of cell growth repression by CG-1521 and TSA, the effects of the two HDACi on cell cycle progression and apoptosis were assessed by flow cytometry. Treatment of SUM149PT cells with CG-1521 for 48 h, results in the accumulation of cells in the G1 phase of the cell cycle with a concomitant reduction in the G2/M phase cell population (Fig.?2A). In contrast, CG-1521 causes accumulation of SUM190PT cells in the G0/G1 phase accompanied by the almost complete loss of cells in S phase (Fig.?2C). The effect of TSA on SUM149PT cells appears to be concentration dependent since 100 nM TSA induces a decrease in G1 with a corresponding increased accumulation of cells in S phase (Fig.?2B). In contrast, TSA at doses 250 nM causes a substantial increase in G2/M accumulation and concomitant decrease in S phase accumulation. In SUM190PT cells, TSA causes a marked increase in G1 accumulation with a significant decrease in the proportion of cells in S phase (Fig.?2D). The effects of CG-1521 and TSA on cell cycle progression are not affected by the absence or presence of E2 in either cell TG100-115 line. Open in a separate window Figure?2. Inhibition of cell cycle progression by CG-1521 and TSA in IBC cells. SUM149PT cells (A and B) and SUM190PT cells (C and D) were treated with indicated doses of CG-1521 (A and C) or TSA (B and D) in the absence or presence of 10 nM E2 for 48 h. Cell cycle kinetics were measured by flow cytometry using propidium iodide staining as described in Methods. SUM149PT cells were treated with 7.5 M CG-1521 (A) or 100 nM or 250 nM TSA (B). SUM190PT cells were treated with 5 M CG-1521 (C) or 1 M TSA (D) for 48 h. For SUM149PT cells, TG100-115 red, G1; dark pink, S phase; light pink, G2/M phase. For SUM190PT cells, dark blue, G1; medium blue, S phase; light blue, G2/M phase. Results represent the mean of three experiments. The error bars are omitted for clarity. The increased levels of DNA fragmentation in both SUM149PT and SUM190PT cells in the absence or presence of E2 (Fig.?3A and TG100-115 C) indicates CG-1521 induces apoptosis, although the SUM190PT cells are more sensitive to CG-1521 compared with SUM149PT cells. In contrast, the SUM149PT cells are highly sensitive while the SUM190PT cells are relatively resistant to TSA treatment (Fig.?3B and D). However, at doses greater than 250 nM, TSA appears to rapidly obliterate SUM149PT cells, leaving too few cells to determine whether there is evidence of DNA fragmentation (data not shown). Open in a separate window Figure?3. Induction of DNA fragmentation by CG-1521 and TSA in IBC cells. SUM149PT cells were treated with 7.5 M CG-1521 (A) or 100 nM TSA (B); SUM190PT cells were treated Rabbit Polyclonal to MARCH3 with 5 M CG-1521 (C) or 1 M TSA (D) in the absence or presence of 10 nM E2 for 48 h. The percentage of cells displaying fragmented DNA was measured using Apo-BrdU staining as described in TG100-115 Methods. Results represent the mean ( SD) from three independent experiments. Comparisons between different treatment groups were analyzed using one-way ANOVA; differences were considered significant if < 0.05 (*), NS, not significant. Effect of CG-1521 and TSA on morphology of IBC cells To examine the effects of CG-1521 and TSA on the cell biology of IBC cells, SUM149PT and SUM190PT cells were treated with the HDACi and examined by confocal microscopy to visualize the actin cytoskeleton and acetylated -tubulin. CG-1521 treatment of SUM149PT cells does not induce a dramatic change in the actin cytoskeleton, which is diffusely distributed throughout the cell with a distinct localization within the plasma membrane. However in dividing SUM149PT cells, CG-1521 prospects to the formation of extended midbody constructions.