Heterogeneity of HDAC3 expression and its modulation by valproic acid in glioblastoma cells.

Abstract

e13038 Background: Glioblastoma (gbm) is a devastating malignancy and a therapeutic challenge. Our group and others have observed long-term gbm survivors treated with valproic acid (VPA), a histone deacetylase (HDAC) inhibitor. While in vitro studies of HDAC inhibitors have been promising, clinical trials and retrospective analyses have been disappointing. We hypothesize that HDAC3 may be a more specific target or treatment marker for gbm. Since limited data is available pertaining to HDAC3 and its modulation in gbm, our goals were to: 1) develop methods for determining HDAC3 expression and localization in gbm cells, 2) study HDAC3 heterogeneity among different gbm cell lines, including one in low passage, and 3) test the effect of VPA treatment on HDAC3 expression of gbm cells in vitro. Methods: Three established gbm cell lines (U-87, U-251 and E297), were cultured using standard technique. HeLa cells were used as the positive HDAC3 control. Cells were stained using 2 different antibodies, and analyzed using immunofluoresence microscopy (IFM), and Western blotting (with β-actin standards). IgG was used for negative isotype controls. Cell counts, viability, and H&E staining for changes in morphology were also recorded. Lysates for Western blots were made from whole cells, nuclei, and cytoplasmic preparations. Band quantification was performed using the ImageJ software. Cells in exponential growth were treated for 48 hours with 10 mM VPA, and compared to untreated cells. Results: Untreated cells studied for HDAC3 using IFM showed nuclear and some cytoplasmic staining in all 4 lines, which varied according to cell type and morphology. Western blot analysis confirmed the variability of HDAC3 expression seen by IFM. The effect of VPA treatment on gbm HDAC3 expression (cytoplasmic and nuclear) was seen to be highly variable, ranging from marked down-modulation in some gbm cells, to minimal effect in others. Conclusions: These data provide the groundwork for studying HDAC3 levels in patients undergoing therapy with HDAC inhibitors. The effect of VPA on the HDAC3 expression of gbm cells is heterogeneous; individualized assessment of such effect will be necessary in delineating any subset of patients that may respond to VPA treatment.