We then demonstrated that the effects of TSA on induction of gene expression are operative in supplemental medulloblastoma cell lines. TSA treatment method induced expression of p21 and RASSF1 in D283 and Daoy medulloblastoma cell lines and in MB100 key cell cultures. Both p21 and RASSF1 are previ ously identied as genes induced by TSA. We upcoming analyzed the func tional signicance of the up regulated genes by mapping them to different pathways applying the PANTHER classi cation procedure. Of the 714 genes up regulated not less than twofold, 106 mapped to 68 known signaling pathways. Predominant in these have been pathways involved in carcinogenesis this kind of as angiogen esis, apoptosis, and much more specically, the Ras, p53, and Wnt signaling cascades. Even though many within the genes have not been previously associated with medulloblastoma, pathways regarded to be involved in medulloblastoma pathogenesis, such as sonic hedgehog signaling, as well as EGF and IGF receptor tyrosine kinase signaling, have been also identied through the PANTHER analysis.
Furthermore, quite a few TSA induced genes function dig this in cerebellar build ment or probably in medulloblastoma pathogenesis. One example is, PAX family gene expression has previ ously been related with medulloblastoma. Similarly, Notch mediated signaling was recently linked with tumor formation in medullo blastoma mouse versions. DKK1 Is Down regulated in Medulloblastoma and Induced by HDAC Inhibition Our objective was to identify genes epigenetically silenced by histone deacetylation which might be reversibly induced by TSA and therefore are candidate tumor suppressor genes. Of 714 genes up regulated on TSA therapy, we observed sev eral genes previously proven to suppress tumor development in other cancers. Among these genes was DKK1, a Wnt antagonist that affects cell development.
We examined adjustments in DKK1 expression on TSA remedy in three patient derived key medulloblastoma cell lines and 1 immortalized cell line with respect to usual cerebellum by reverse selleck Screening Library transcriptase PCR. DKK1 expression was signicantly down regulated in all instances and improved on TSA treatment method. To lengthen these ndings to medulloblastoma tumors, we in contrast DKK1 expression in 10 patient tissue samples relative to regular cerebellum by RT PCR. When in comparison to regular cerebellum, all 10 samples expressed 80% less DKK1. Examination of vari ance conrmed that this difference was statistically sig nicant. Histone Acetylation Regulates DKK1 Expression in Medulloblastoma To even more validate the role of histone tail modications as an epigenetic silencing mechanism for DKK1 in medulloblastoma, we carried out ChIP utilizing antibodies against acetylated histones H3 in the Lys9 position. Con sistent with our earlier benefits, TSA treatment enhanced vefold the histone acetylation inside the promoter region of DKK1. These data recommend that reversal of histone deacety lation by TSA was sufcient to allow DKK1 gene expres sion in medulloblastoma cells.