There are two major CBFb isoforms that are highly conserved in mammals

ts Glioma Stem-Like Cell Proliferation doi: 10.1371/journal.pone.0080714.g004 an HDACi to prevent in vitro deacetylation, GTA increased 6807310 acetylation of several proteins. The acetylation was specific since no increase in actin acetylation was detected. Inasmuch as acetylation is a dynamic process, it is possible that proteins acetylated by GTA could have become deacetylated either in culture or subsequent to extraction in triton lysis buffer. Therefore, cells were treated with 0.25% GTA in the absence or presence of the HDACi SAHA for 24 hours, extracted with RIPA buffer to increase histone protein extraction, and analyzed by western blot analysis. Not unexpectedly, there was 10 GTA Inhibits Glioma Stem-Like Cell Proliferation doi: 10.1371/journal.pone.0080714.g005 a significant increase in the number of acetylated proteins in SAHA treated cells. Even in the absence of SAHA, GTA 16707462 increased protein acetylation, including several within the molecular weight range for histones H3 and H4. Preliminary mass spectrometry analysis indicates increased acetylation of histone H4K8, H4K12, and H4K16 as well as other proteins involved in cell cycle regulation. Moreover, recent microarray data revealed that GTA significantly decreased expression of several histone isoforms in OG33 cells, which expresses a novel nuclear restricted ASPA isoform, but not OG35 cells expressing only full length ASPA, even though GTA induced comparable cytostatic growth arrest in both cells. Collectively, our data suggest that GTA exerts growth inhibitory effects via an epiFD&C Green No. 3 chemical information genetic mechanism without promoting differentiation and that cells with the novel ASPA isoform may utilize the acetate differently than those only expressing fulllength ASPA. Discussion Due to the decreased incidence and better prognosis of oligodendroglioma relative to GBM, few studies have 11 GTA Inhibits Glioma Stem-Like Cell Proliferation doi: 10.1371/journal.pone.0080714.g006 12 GTA Inhibits Glioma Stem-Like Cell Proliferation doi: 10.1371/journal.pone.0080714.g007 13 GTA Inhibits Glioma Stem-Like Cell Proliferation doi: 10.1371/journal.pone.0080714.g008 endeavored to generate oligodendroglioma cell lines. Although OG33 and OG35 cells were derived from primary oligodendroglioma tumors using a well-established protocol for the generation of GSCs, these cells exhibit only some GSC and oligodendroglioma features. Although the OG cells exhibit 1p/19q LOH, O6methylguanine-methyltransferase promoter hypermethylation, self-renewal, and form aggressive orthotopic tumors, they express wild-type IHD1/2. Since all four oligodendroglioma lines used in the study expressed wild-type IDH1/2, it is possible that cells harboring IDH mutations cannot be propagated in vitro because cell culture selects for the more aggressive wild-type IDH1/2 cells. In fact, cells must be transfected to express mutant IDH1/2 to study their function. Interestingly, this study showed that NAAG was reduced 50-fold and 8.3-fold in HOG cells expressing IDH1 and IDH2 mutants, respectively. Therefore, it would be intriguing to assess whether ASPA protein levels, ASPA subcellular localization, or GTA-mediated growth arrest are altered in cells bearing IDH1/2 mutations. Another distinctive feature of the OG cells is their lack of differentiation into astrocytes or mature MBP-positive oligodendrocytes, as would be expected of OPCs, a proposed glioma cell of origin. Oligodendrogliomas exhibit a proneural genetic signature and the OG cells expressed