Summary: We present easyFRAP, a versatile tool that assists quantitative and qualitative analysis of fluorescence recovery after photobleaching (FRAP) data. The user can handle simultaneously large data sets of raw data, visualize fluorescence recovery curves, exclude low quality data, perform data normalization, extract quantitative parameters, perform batch analysis and save the resulting data and figures for further use. Our tool is implemented as a single-screen Graphical User Interface (GUI) and is highly interactive, as it permits parameterization and visual data quality assessment at various points during the analysis. Availability: easyFRAP is free software, available under the General Public License (GPL). Executable and source files, supplementary material and sample data sets can be downloaded at: ccl.med.upatras.gr/easyfrap.html. Contact:lygerou@med.upatras.gr; rapsoman@upatras.gr Supplementary information: Supplementary data are available at Bioinformatics online.

S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in G₁, and passage through S-phase removes the license to replicate. In fission yeast, Cdc6/18 and Cdt1, two factors required for licensing, are central to ensuring that replication occurs once per cell cycle. We show that the human Cdt1 homologue (hCdt1), a nuclear protein, is present only during G₁. After S-phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G₂. hCdt1 can associate with the DNA replication inhibitor Geminin, however these two proteins are mostly expressed at different cell cycle stages. hCdt1 mRNA, in contrast to hCdt1 protein, is expressed in S-phase-arrested cells, and its levels do not change dramatically during a cell cycle, suggesting that proteolytic rather than transcriptional controls ensure the timely accumulation of hCdt1. Consistent with this view, proteasome inhibitors stabilize hCdt1 in S-phase. In contrast, hCdc6/18 levels are constant through most of the cell cycle and are only low for a brief period at the end of mitosis. These results suggest that the presence of active hCdt1 may be crucial for determining when licensing is legitimate in human cells.

The accurate execution of DNA replication requires a strict control of the replication licensing factors hCdt1 and hCdc6. The role of these key replication molecules in carcinogenesis has not been clarified. To examine how early during cancer development deregulation of these factors occurs, we investigated their status in epithelial lesions covering progressive stages of hyperplasia, dysplasia, and full malignancy, mostly from the same patients. Abnormal accumulation of both proteins occurred early from the stage of dysplasia. A frequent cause of unregulated hCdc6 and hCdt1 expression was gene amplification, suggesting that these components can play a role per se in cancer development. Overexpression of hCdt1 and hCdc6 promoted rereplication and generated a DNA damage response, which activated the antitumor barriers of senescence and apoptosis. Generating an inducible hCdt1 cellular system, we observed that continuous stimulus by deregulated hCdt1 led to abrogation of the antitumor barriers and resulted in the selection of clones with more aggressive properties. In addition, stable expression of hCdc6 and hCdt1 in premalignant papilloma cells led to transformation of the cells that produced tumors upon injection into nude mice depicting the oncogenic potential of their deregulation. [Cancer Res 2007;67(22):10899–909]

Multiciliated cells are abundant in the epithelial surface of different tissues, including cells lining the walls of the lateral ventricles in the brain and the airway epithelium. Their main role is to control fluid flow and thus defects in their differentiation were implicated in many human disorders such as hydrocephalus, accompanied by defects in adult neurogenesis and mucociliary disorder in the airway system. Here we show that Mcidas, which was mutated in human mucociliary clearance disorder and GemC1/Lynkeas, previously implicated in cell cycle progression, are key regulators of multiciliated ependymal cells generation in the brain. Overexpression and knock down experiments show that Mcidas and GemC1/Lynkeas are sufficient and necessary for cell fate commitment and differentiation of radial glial cells to multiciliated ependymal cells. Furthermore, we show that GemC1/Lynkeas and Mcidas operate in hierarchical order, upstream of Foxj1 and c-Myb transcription factors, known regulators of ependymal cell generation, while Notch signaling is inhibiting their function. Our results suggest that Mcidas and GemC1/Lynkeas are key players for the generation of multiciliated ependymal cells of the adult neurogenic niche.