These proteins included c-Myc, SOX2, and OCT4, whose expression is related with reprogramming of somatic cells to a stem cell phenotype. Expression of c-Myc, SOX2, and OCT4 in cells expressing wild-type, EMD 1214063 C3�CC10, ��C4�CC7, and ��37(C286,295A) of CD44s and Cont-shRNA increased during sphere formation in suspension culture (Figure 4C). The similar results were also shown in HCT-116 and DLD-1 cells (Supplementary Figure S5A). To confirm protein expression, spheres were immunostained for SOX2 and OCT4. After 12 days in suspension, we observed only low-level cytoplasmic staining for SOX2 and OCT4 in cells expressing wild-type CD44s maintained as subconfluent monolayers (WT/AD) and spheres expressing the CD44s(NLS) mutant (NLS mut/SPH) (Figure 4D).

In contrast, nuclear immunostaining for SOX2 and OCT4 became evident in spheres expressing wild-type CD44s (WT/SPH). This nuclear immunostaining was also found in spheres expressing wild-type, C3�CC10, ��C4�CC7, and ��37(C286,295A) of CD44s and Cont-shRNA (data not shown). Figure 4 CD44-expressing cells are reprogrammed into stem-like cells after the suspension culture. (A) Validation by qChIP-PCR of putative nuclear CD44 target genes in HT29/CD44+ spheres. A ChIP assay was performed with chromatin from HT29/CD44+ … Multipotential stem cells have been characterized as a side population based on their properties compared with the main population of cells present (Challen and Little, 2006). Also, these side-population properties are shared by a small subset of cells in tumours, referred to as CSCs, that can recapitulate the tumour heterogeneity in xenographic transplants (Challen and Little, 2006).

Because of the reexpression of stem cell-related genes (Figure 4D) and the stable changes in morphology seen in sphere-derived cells (Figure 1E), we wondered if sphere formation might be reprogramming the CD44-expressing cancer cells to generate side-population cells with properties of CSCs. We began by examining HT29/CD44?/CD44-myc cells maintained as subconfluent monolayers (AD) and cells derived from spheres (SPH) in Hoechst dye�Cexclusion assays. We found that CD44s/Mock maintained as subconfluent monolayers (AD) and cells derived from nuclear CD44s/STAT3 signalling-defective spheres (CD44s(NLS mut)/Mock, CD44s/STAT3(K685R), CD44s/STAT3(Y705F, K685R), CD44s/STAT3-shRNA, CD44s/p300-shRNA, and CD44s/HDAC1) did not exclude Hoechst dye (Figure 4E).

However, ~10% of sphere cells expressing CD44s/Mock, CD44s/STAT3(Y705F), and CD44s/Cont-shRNA were Hoechst negative. The similar results were also shown in HCT-116 and DLD-1 cells (Supplementary Figure S5B�CD). Indeed, properties such as anchorage independence, which are thought to be a hallmark of transformed cells, have recently been described as a property of stem cells Brefeldin_A (Reynolds and Weiss, 1996; Weiss et al, 1996; Dontu et al, 2003).