Anti-PROM1 monoclonal antibody Made to order

Medulloblastoma is the most frequent type of embryonal tumour in the paediatric population. The disease progression in patients with this tumour may be connected with the presence of stem/tumour-initiating cells, but the precise source and characteristics of such cells is still a subject of debate. Thus, we tried to analyse biomarkers for which a connection with the presence of stem/tumour-initiating cells was suggested. We evaluated the transcriptional level of the ATOH1, FUT4, NGFR, OTX1, OTX2, PROM1 and SOX1 genes in 48 samples of medulloblastoma and analysed their usefulness in the prediction of disease outcome. The analyses showed a strong correlation of PROM1, ATOH1 and OTX1 gene expression levels with the outcome (p a parts per thousand currency signaEuro parts per thousand 0.2). On the basis of the multivariate Cox regression analysis, we propose a three-gene model predicting risk of the disease, calculated as follows: . Survival analysis revealed a better outcome among standard-risk patients, with a 5-year survival rate of 65 %, compared to the 40 % rate observed among high-risk patients. The most promising advantage of such molecular analysis consists in the identification of molecular markers influencing clinical behaviour, which may in turn be useful in therapy optimization.

Tumor stem cells, postulated to be the source cells for malignancies, have been identified in several cancers using cell-surface expression of markers including CD133, a pentaspan membrane protein. CD133+ve cells form neurospheres, exhibit self-renewal and differentiation, and are tumorigenic. However, despite its association with stem cells, a causal relationship of CD133 to tumorigenesis remains to be defined. Hypothesizing that specific epigenetic and transcription factors implicated in driving the stem cell state may concurrently regulate CD133 expression in stem cells, we analyzed the structure and regulation of CD133 promoter in glioma stem cells and glioma cell lines. Initially, a minimal promoter region was identified by analyzing the activity of CD133 promoter-driven luciferase-expressing 5'-and 3'-deletion-constructs upstream of the transcription start site. This region contained a CpG island that was hypermethylated in CD133-ve glioma stem cells (GSC) and glioma cells but unmethylated in CD133+ve ones. Of several predicted TF-binding sites in this region, the role of tandem Sp1 (-242 and -221) and two Myc (-541 and -25)-binding sites were examined. Overexpression of Sp1 or Myc increased CD133 minimal promoter-driven luciferase activity and CD133 levels in GSC and in glioma cell line. Mithramycin, a Sp1 inhibitor, decreased minimal promoter activity and downregulated CD133 levels in GSC. Gel-shift assays demonstrated direct binding of Sp1 to their predicted sites that was competitively inhibited by oligonucleotide-binding-site sequences and supershifted by anti-Sp1 confirming the interaction. Sp1 and Myc-antibody chromatin immunoprecipitation (ChIP) analysis in GSC showed enrichment of regions with Sp1 and Myc-binding sites. In CD133-ve cells, ChIP analysis showed binding of the methyl-DNA-binding proteins, MBD1, MBD2 and MeCP2 to the methylated CpG island and repression of transcription. These results demonstrate that Sp1 and Myc regulate CD133 transcription in GSC and that promoter methylation and methyl-DNA-binding proteins cause repression of CD133 by excluding transcription-factor binding.