Winged helix (wH) domains, also termed winged helix-turn-helix (wHTH) domains, are widespread in all kingdoms of life and have diverse roles. In the context of DNA binding and DNA modification sensing, some eukaryotic wH domains are known as sensors of non-methylated CpG. In contrast, the prokaryotic wH domains in DpnI and HhiV4I act as sensors of adenine methylation in the 6mApT (N6-methyladenine, 6mA, or N6mA) context. DNA-binding modes and interactions with the probed dinucleotide are vastly different in the two cases. Here, we show that the role of the wH domain as a sensor of adenine methylation is widespread in prokaryotes. We present previously uncharacterized examples of PD-(D/E)XK—wH (FcyTI, Psp4BI), PUA—wH—HNH (HtuIII), wH—GIY-YIG (Ahi29725I, Apa233I), and PLD—wH (Aba4572I, CbaI) fusion endonucleases that sense adenine methylation in the Dam+ Gm6ATC sequence contexts. Representatives of the wH domain endonuclease fusion families with the exception of the PLD—wH family could be purified, and an in vitro preference for adenine methylation in the Dam context could be demonstrated. Like most other modification-dependent restriction endonucleases (MDREs, also called type IV restriction systems), the new fusion endonucleases except those in the PD-(D/E)XK—wH family cleave close to but outside the recognition sequence. Taken together, our data illustrate the widespread combinatorial use of prokaryotic wH domains as adenine methylation readers. Other potential 6mA sensors in modified DNA are also discussed.

Winged helix (wH) domains, also termed winged helix-turn-helix (wHTH) domains, are widespread in all kingdoms of life, and have diverse roles. In the context of DNA binding and DNA modification sensing, some eukaryotic wH domains are known as sensors of non-methylated CpG. In contrast, the prokaryotic wH domains in DpnI and phi.HhiV4I act as sensors of adenine methylation in 6mApT (6mA = N6mA) context. DNA binding modes and interactions with the probed dinucleotide are vastly different in the two cases. Here, we show that the role of the wH domain as a sensor of adenine methylation is widespread in prokaryotes. We present previously uncharacterized examples of PD-(D/E)XK—wH (FcyTI, Psp4BI), PUA—wH—HNH (HtuIII, Hsa13891I), wH—GIY-YIG (Ahi29725I, Apa233I) and PLD—wH (Aba4572I, CbaI) fusion endonucleases that sense adenine methylation in the Dam G6mATC, and possibly other, slightly more relaxed contexts. Representatives of the wH domain endonuclease fusion families with the exception of the PLD—wH family could be purified, and an in vitro preference for adenine methylation in the Dam context could be demonstrated. Like most other MDREs, the new fusion endonucleases except those in the PD-(D/E)XK—wH family cleave close to, but outside the recognition sequence. Taken together, our data illustrate the widespread combinatorial use of prokaryotic wH domains as adenine methylation sensors.

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.

Cancers account for 85% of renal tumors. In Poland renal cancer is diagnosed in almost four thousands patients every year and two thousands of them dies. The most common subtype of renal cancer is clear cell renal cell carcinoma (ccRCC), which accounts for 80-90% of all renal cancer cases. ccRCC is resistant to chemo- and radiotherapy. More and more data suggest that tumor growth is a result of proliferation and differentiation of a small population of cells called cancer stem cells (CSC). CSCs are responsible for tumor progression and for the resistance to chemo- and radiotherapy. This publication covers the role the CSCs and their origin in renal cell carcinoma, with particular emphasis on clear cell subtype.

Raki stanowią 85% guzów nerki. W Polsce każdego roku raka nerki rozpoznaje się u prawie czterech tysięcy osób, a ponad dwa tysiące z nich umiera. Znanych jest kilka rodzajów raka nerki, spośród których najczęściej występującym jest rak jasnokomórkowy (ang. clear cell Renal Cell Carcinoma; ccRCC), który występuje w 80-90 proc. wszystkich przypadków raka nerki. ccRCC należy do nowotworów opornych na leczenie chemiczne i radioterapię. Coraz więcej danych wskazuje iż masa nowotworu powstaje w efekcie podziałów i różnicowania niewielkiej liczby komórek zwanych komórkami macierzystymi nowotworu (komórkami inicjującymi guz ang. cancer stem cells - CSC). CSC są nie tylko odpowiedzialne za rozwój nowotworu, ale także za wznowę, progresję choroby i jej oporność na chemio- i radioterapię. W pracy omówiono pochodzenie CSC w raku nerki, ze szczególnym uwzględnieniem podtypu jasnokomórkowego.

Cancer stem cells (CSCs) are defined as a fraction of tumor cells having a clonogenic ability, expressing stemness transcriptional factors such as Nanog, Nestin, Oct4 and Pax2. CSCs for the first time identified in leukemia, have also been recently described in renal cancer (RCC). These cells can form 3D spheres, that differentiate in vitro into endothelial and epithelial cells, and induce tumors in SCID mice. Isolation techniques and CSCs studies use the specific properties of these cells to separate them from tumor mass or cell culture. Today, methods for the isolation of RCC CSCs that are based on affinity to the surface antigen CD105 (endoglin) are known. Similarly, CD133(+) cells in RCC are most likely CSCs as well as CXCR-4 cells. Similarly to the CSC isolation, the expression of intracellular markers is used, including ALDH1, DNAJB8 and PIK3R1. These isolation methods allow CSCs to be harvested from primary and stabilized cell line cultures as well as cell culture after isolation from xenografts and in the presence of drugs. An additional method of CSC isolation is the isolation of cells called side population (SP) with the use of Hoechst 33342 dye or rhodamine (Rh123). SP cells display high ALDH1 activity. The parent phenotype can also be induced by manipulating the composition of the cell culture medium. Similarly, the cultivation of 3D cell spheres are conditions even closer to in vivo and promote CSC growth. The 2D cell culture with reduced concentration of growth factors has similar properties. The huge diversity of markers and methods of RCC-CSC insulation contributes to difficulties in determining the optimal method of isolation and requires further optimization.

e16091 Background: Tumor initiating cells represent a minor cell subpopulation that is resistant to therapies and has cancer stem cell characteristics including self-renewal, multiple lineage differentiation, tumor initiation and cancer dissemination potential. Endoglin has been shown as renal cell cancer tumor initiating cells surface marker. Methods: Endoglin expressing subpopulation of cells was isolated from ACHN (metastatic RCC) and CAKI-2 (primary RCC) cell lines under FACSAriaII based protocol. Agilent human GE 4x44K v2 microarrays and GeneSpring GX 13.0 package were used for gene expression analysis. (Sponsored: TEAM/2010-6/8 FNP). Results: In comparison to normal renal proximal tubule cells in endoglin expressing subpopulation of cells from metastatic and primary tumors - 2324 and 1346 genes were overexpressed while 2763 and 1614 genes were down-regulated, respectively. 1411 genes were commonly deregulated in metastatic and primary tumor derived endoglin positive cells. TGF-β, Wnt/β-catenine signalling and Epithelial-Mesenchymal Transition pathway were deregulated in renal cell cancer tumour initiating cells. TGFB1, ERBB2, and TNF are transcriptional regulators in these cells. Gene Set Enrichment Analysis has shownRap1, PI3K-Akt and Hippo signaling pathways as significantly deregulated in endoglin positive renal cell cancer cells. Genes expressed were shown to be primarily located in TP53, cytokine and kinase networks. Conclusions: Tumor initiating cells share deregulated pathways with typical normal stem cells, however rely on distinct signalling networks to maintain stemness and possibly to contribute to the progression of disease and metastasis. Renal cell cancer tumor initiating cells may also represent novel treatment target and their gene expression profile could be used as initial data for new functional and drug design studies.

BACKGROUND:Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells-stem cell-like cancer cells (SCLCCs)-which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin.MATERIALS AND METHODS:Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers-CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent's human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis.RESULTS:Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-β, Wnt/β-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells.CONCLUSIONS:All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design.

In recent years, cancer stem cells (CSCs)/tumor initiating cells (TICs) have been identified inside different tumors. However, currently used anti-cancer therapies are mostly directed against somatic tumor cells without targeting CSCs/TICs. CSCs/TICs also gain resistance to chemotherapies/radiotherapies. For the development of efficient treatment strategies, choosing the best method for isolation and characterization of CSCs/TICs is still debated among the scientific community. In this review, we summarize recent data concerning isolation techniques for CSCs using magnetic cell sorting and flow cytometry. The review focuses on the strategies for sample preparation during flow cytometric analysis, elaborating biomarkers such as CXCR4, CD105, and CD133. In addition, functional properties characteristic of CSCs/TICs using side population selection through Hoechst 33342 dye, aldehyde dehydrogenase 1, dye-cycle violet, and rhodamine 123 are also discussed. We also include a special focus on enriching CSCs/TICs using three-dimensional cell culture models such as agarose–agarose microbeads and sphere formation.