Subsequently, protein concentrations were determined for the fractions using the BCA protein assay. Equivalent amounts of protein were loaded in each lane. Following trans fer to nitrocellulose, the blots http://www.selleckchem.com/products/INCB18424.html were first stained with Pon ceau Red to assess protein loading, and subsequently probed with the following antibodies phospho Hsp27S15 and Hsp27. Blots were cut and reprobed sequentially, and visualized with ECL reagents and exposure to X ray film. Developed films were subsequently digitized and densitometrically analyzed with a cyclone ChemiImager and AlphaEase software. Digital images of the blots were used to make composite figures with Adobe Photoshop graphics software. Background Gliomas, the most common brain tumor, are currently classified as astrocytic, ependymal, oligodendroglial and choroid plexus tumors.

Among astrocytic tumors, gliob lastoma is the most lethal primary malignant brain tumor. Although considerable progress has been made in its treatment, the clinical prognosis associated with this tumor remains poor. Histone deacetylases have recently become rec ognized as a promising target for cancer therapy, includ ing for the treatment of glioblastomas. Together with histone acetyltransferases, HDACs are responsible for chromatin packaging, which influences the transcrip tion process. In general, increased levels of acetylation are associated with increased transcrip tional activity, whereas decreased acetylation levels are associated with repression of transcrip tion. HDACs are classified into 4 major categories based on their homology to yeast HDACs, including structure and cellular localization.

Class I and class II HDAC proteins share a common enzy matic mechanism that is the Zn catalyzed hydrolysis of the acetyl lysine amide bond. Human class I HDACs includes HDAC1, 2, 3, and 8, which are enzymes simi lar to the yeast transcriptional regulator Rpd3, generally localized to the nucleus. These enzymes are ubiqui tously expressed and seems to function as a complex with other proteins. HDAC1 and 2 only show activity within a protein complex, which consists of proteins necessary for modulating their deacetylase activity and DNA binding, and the recruit ment of HDACs to gene promoters. Wilson AJ et al. have suggested that multiple class I HDAC members are also involved in repressing p21 and that the growth inhib itory and apoptotic effects induced by HDAC inhibitors are probably mediated through the inhibition of multiple HDACs.

Class II HDACs includes HDAC4, 5, 6, 7, 9a, Brefeldin_A 9b, and 10, which are homologous to yeast Hda1. These class II enzymes can be found in the nucleus and cytoplasm, sug gesting potential extranuclear functions by regulating the acetylation status of nonhistone substrates. HDAC members of class II are abundantly expressed in skeletal muscle, heart, brain, tissues with low levels of mitotic activity.