Results P5 exhibits the highest activity among the five putative promoters of CD133 gene To analyze the transcriptional machinery of CD133 gene, we used a human colon carcinoma cell line Caco 2 and a human synovial sarcoma cell line Fuji, because both cell lines express selleck inhibitor abundant CD133 transcripts and proteins, and also CD133 Caco 2 cells are reported to be able to generate tumors following transplantation in mice, whereas CD133 cells are not. Our previous studies showed that Caco 2 expresses the transcripts containing all variants of exon 1, and Fuji expresses only exons 1A, 1B, 1C, and 1E. Immunoblotting and RT PCR analysis confirmed that the expression levels of CD133 mRNA and protein in Caco 2 cells are higher than those in Fuji cells.

FACS analysis using anti CD133 antibody revealed that both cell lines exhibit the cellular heterogeneity for CD133 expression, as the CD133 positive fraction is 88. 9% and 18. 2% in Caco2 and Fuji cells, respectively. Luciferase assay demonstrated a remarkable activation of P5 and a modest acti vation of P1, and no significant enhancement of P2 and P3 was detected Inhibitors,Modulators,Libraries in both cells. A modest activation of P4 was observed only in Caco 2 cells, which probably contri butes to the expression of exon 1D containing transcripts. Ets binding motifs are required for CD133 P5 activity To determine the minimal region essential for P5 pro moter activity, we generated a series of deletion mutants of P5 promoter and found that deletion to 98 holds a similar luci ferase activity to the full length promoter, but the further deletion to 25 leads to a significant reduction of activity, suggesting that the region between 98 and 25 contains minimal elements required for CD133 transcription through P5 promoter.

The region between 98 to 25 contains two consensus binding motifs of Ets family proteins GGAAG. To determine whether these Ets motifs are necessary for P5 activity, we introduced a substitutional mutation altering two nucleotides Inhibitors,Modulators,Libraries of Ets core sequence GGAA to TTAA, into each or both of the Ets binding sites, designated as pGL3enh P5 98mEBS 1, 2, and 1 2. Introduction of Inhibitors,Modulators,Libraries the mutation at EBS 1 moderately decreased P5 promo ter activity and the mutation at EBS 2 remarkably decreased the activity. These results indi cated that the two Ets binding motifs are required for the P5 activity, and that in particular, mutation of EBS 2 resulted in the greatest reduction of P5 activity.

Specific binding of nuclear factors Inhibitors,Modulators,Libraries to an Ets motif 2 in CD133 P5 Next, to investigate the specific Inhibitors,Modulators,Libraries transcription factor which binds to EBS between 98 to 25 of P5, EMSA was performed using nuclear proteins extracted from Caco 2 and Fuji cells. A 25 bp double stranded oli goDNA containing 3-deazaneplanocin A HCl putative Ets binding sites was synthesized as probes or as unlabeled competitors.