Treatment of Fuji cells with U0126 led to the marked inhibition of P5 activity. No cell toxicity was observed concerning mor phology and selleck chemical growth of both cell lines under the conditions of this experiment. Consis tently, U0126 also markedly decreased the expression of CD133 protein in Fuji cells, indicating that the MEK/ERK signaling is implicated in P5 mediated CD133 expression. In contrast, Inhibitors,Modulators,Libraries the same concentration of U0126 could not affect P5 activity and expression of CD133 in Caco 2 cells, suggesting that another pathway could regulate Ets mediated P5 transcription in Caco 2 cells. However, it remains possible that the ERK path way might simultaneously regulate other promoters. In fact, ERK inhibition decreased the expression of exon 1B and 1D and 1E containing CD133 mRNA, but increase exon 1C containing one.

These data indicate cell type specific regula tion of CD133 gene expression by the ERK pathway. Inhibition of MEK/ERK pathway abolishes side population in Caco 2 cells To assure the relevance of the Inhibitors,Modulators,Libraries ERK pathway to stem like characteristics, the effect of U0126 on the amount of SP was assessed by flow cytometric analysis. The SP frac tion in tumor cells has been known to define the popu lation containing stem like cells, which highly express ATP binding cassette transporters to efflux both Hoechst dye and chemotherapeutic agents, and to have a high capacity to form tumor xenografts in mice. In our experiments, the side population represented approximately 2% in the Caco 2 cell line. Treatment with verapamil, an inhibitor of the ABC transporters, completely ablated this popula tion.

In contrast, no distinct SP was visible in the Fuji cell line. Treatment of Caco 2 with U0126 dramatically reduced the SP frequency to 0. 15%. This result emphasized our con clusion that ERK is a key molecule in the signal trans duction to maintain stem like features in tumor Inhibitors,Modulators,Libraries cells. Ets2 increases CD133 Inhibitors,Modulators,Libraries mRNA levels in human astrocytes, but cannot confer tumorigenicity To examine whether Ets factor could increase CD133 expression and confer tumorigenicity in normal cells, we established the immortalized human astrocytes overex pressing Ets2. The increase of CD133 mRNA expression was detected in NHA/TSE2 compar ing to NHA/TS cells and its effect was strongly Inhibitors,Modulators,Libraries enhanced by the treatment with demethylating agent 5 Aza dC and histone deacetyltransferase inhibitor TSA.

5Aza dC/TSA treatment has been shown to open the chromosomal region to increase accessibility for transcription factor complexes different to assemble at the promoter and drive gene transcription. However, the elevated protein level of CD133 could not be detected by FACS analysis. Instead, approximately 0. 04% of side population diminished by verapamil was observed in the NHA/TSE2 cells, whereas substantial SP was not visible in NHA/TS cells.