Error bars repre sent the Standard Error of the Mean and each experiment has been completed at least twice www.selleckchem.com/products/CP-690550.html with samples in triplicate. Results Identification of differentially methylated genes in invasive sub populations of cells Individual promoter tiling arrays were performed to analyze global CpG promoter methylation for both non invasive and invasive cell isolates from both LNCaP and DU145. The cells were allowed to invade the Matrigel toward a highly defined media called stem cell media. It was then determined which genes were methylated in the non invasive cells and not in the invasive fraction of cells. This analysis determined that 869 probes were differentially methylated in the non invasive LNCaP fraction compared with the invasive and 1015 for DU145.

A very small subset of 44 overlapping genes was Inhibitors,Modulators,Libraries methylated in the non invasive cells and not in the inva sive population from both of the prostate cancer lines analyzed. These included genes involved in development such as Irx3, Six1 and Sox1, as well as a type III 5 deio dinase, and an embryonic version of myosin. Using the Oncomine database we investigated changes in expression patterns for these methylated targets, and we found a significant associa tion between progression of prostate cancer and metas tasis with expression of a number of genes including G protein, beta 1 subunit, retinoblastoma binding protein 8, secretogranin III and Sox1. Albeit Inhibitors,Modulators,Libraries a number of these proteins have been shown to play a role Inhibitors,Modulators,Libraries in cancer, we chose to investigate the role of Sox1 in our model since it is very homolo gous to the induced pluripotent stem cell regulator Sox2, and has been shown to play a role in progression of lung and nasopharyngeal cancer.

We also chose to investigate bone marrow tyrosine kinase gene in chromosome X protein since Inhibitors,Modulators,Libraries it has been shown to regulate hematopoiesis and play a role in the regulation of prostate cancer. However, from our Oncomine analysis Bmx was not shown to signifi cantly affect prostate cancer metastasis. Verification of methylation array data To verify the results from our methylation specific pro moter tiling arrays, we performed methylation specific PCR where primers were designed around the probe sequences identified from the arrays. Inhibitors,Modulators,Libraries Both Bmx and Sox1 were found to be methylated in the parental LNCaP and DU145 cell lines, representing the non invasive phenotype.

To deter mine if this pattern of methylation correlated with the level of gene expression, real time quantitative PCR was performed. Significant differences in the expression antagonist Bicalutamide of Bmx and Sox1 were seen when comparing the expression in non invasive and invasive cell popula tions in both LNCaP and DU145 cell lines. To further validate the results, immunocytochemistry was performed to analyze differences in protein expres sion between non invasive and invasive cells.