Cancer stem cells (CSCs) have the ability to self-renew similar to normal stem cells. This process is linked with metastasis and resistance to chemotherapy and radiotherapy. In the present study, we constructed an in vitro differentiation model for CSCs. CSCs isolated and proliferated for one passage were maintained as monolayers or spheroid-forming cells with serum included media for differentiation process. Differentiation of adhesion molecules and cellular ultrastructural properties were investigated and compared in both monolayer and spheroid cultures. CD133+/CD44+ cancer-initiating cells were isolated from DU-145 human prostate cancer cell line monolayer cultures and propagated as tumor spheroids and compared with the remaining heterogeneous cancer cell bulk population. Microarray-based gene expression analysis was applied to determine genes with differential expression and protein expression levels of candidates were analyzed by immunohistochemistry. Electron microscopy showed detailed analysis of morphology. TGFβ1 was found to be significantly upregulated in monolayer CSCs. High expression levels of VCAN, COL7A1, ITGβ3, MMP16, RPL13A, COL4A2 and TIMP1 and low expression levels of THBS1, MMP1 and MMP14 were detected when CSCs were maintained as serum-grown prostate CSC spheroids. Immunohistochemistry supported increased immunoreactivity of TGFβ1 in monolayer cultures and VCAN in spheroids. CSCs were found to possess multipotential differentiation capabilities through upregulation and/or downregulation of their markers. TGFβ1 is a triggering molecule, it stimulates versican, Col7A1, ITGβ3 and, most importantly, the upregulation of versican was only detected in CSCs. Our data support a model where CSCs must be engaged by one or more signaling cascades to differentiate and initiate tumor formation. This mechanism occurs with intracellular and extracellular signals and it is possible that CSCc themselves may be a source for extracellular signaling. These molecules functioning in tumor progression and differentiation may help develop targeted therapy.

Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133high/CD44high DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133high/CD44high population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.

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Dishevelled (Dvl) proteins are activated by Wnt pathway stimulation and have crucial roles in the regulation of β-catenin destruction complex. CYLD is a tumor suppressor and a deubiquitination enzyme. CYLD negatively regulates the Wnt/β-catenin signaling pathway by deubiquitinating Dvl proteins. Loss of function and mutations of CYLD were linked to different types of solid tumors. Loss of function in CYLD is associated with Dvl hyper ubiquitination, resulting in the transmission of Wnt signaling to downstream effectors. β-catenin upregulation is observed during disease progression in chronic myeloid leukemia (CML). Deregulated Dvl signaling may be a reason for β-catenin activation in CML; and CYLD may contribute to Dvl deregulation. First, we evaluated mRNA expression in three CML cell lines and mRNA expression of the CYLD gene was found to be present in all (K562, MEG01, KU812). Unlike solid tumors sequencing revealed no mutations in the coding sequences of the CYLD gene. DVL genes were silenced by using a pool of siRNA oligonucleotides and gene expression differences in CYLD was determined by RT-PCR and western blot. CYLD protein expression decreased after Dvl silencing. An opposite approach of overexpressing Dvl proteins resulted in upregulated CYLD expression. While previous reports have described CYLD as a regulator of DVL proteins; our data suggests the presence of a more complicated reciprocal regulatory mechanism in CML cell lines.

Recent studies have described linkage and association between cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene polymorphism and type 1 diabetes mellitus (DM1) in some ethnic populations, but not others. This finding suggests that CTLA-4 gene association with DM1 may be influenced by the racial composition of the population. Thus, it is important to study the polymorphism of the CTLA-4 gene in different ethnic groups. In this case-control association study, the CTLA-4 gene exon 1 A/G polymorphism was analyzed in 48 children with DM1 and 80 healthy controls using polymerase chain reaction-restriction fragment length polymorphism analysis. The possible interaction of the CTLA-4 gene polymorphism with the presence of established genetic markers (HLA-DR genotyping) was also evaluated in 29 patients. The results of the present study do not suggest an association of the known polymorphism in exon 1 of the CTLA-4 gene with DM1 in this Turkish population, and G-allele containing CTLA-4 genotypes were not preferentially associated with age at clinical presentation or with presence of other genetic (HLA-DR3 or -DR4) markers of DM1.