S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in G 1 , 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 G 1 . After S-phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G 2 . 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.

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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 defects in their differentiation are 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 is mutated in human mucociliary clearance disorder, and GemC1 (Gmnc or Lynkeas), previously implicated in cell cycle progression, are key regulators of multiciliated ependymal cell generation in the mouse brain. Overexpression and knockdown experiments show that Mcidas and GemC1 are sufficient and necessary for cell fate commitment and differentiation of radial glial cells to multiciliated ependymal cells. Furthermore, we show that GemC1 and Mcidas operate in hierarchical order, upstream of Foxj1 and c-Myb transcription factors, which are known regulators of ependymal cell generation, and that Notch signaling inhibits GemC1 and Mcidas function. Our results suggest that Mcidas and GemC1 are key players in the generation of multiciliated ependymal cells of the adult neurogenic niche.

Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells. Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin. Over-expression of Cdt1 was shown to predispose cells for malignant transformation. We show here that Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset. Cdt1's inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3-6 h delay over Cdt1, following serum re-addition. Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved. Our data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation. In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium. Cdt1 and geminin levels were compared in primary cells vs. cancer-derived human cell lines. We show that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and that the Cdt1 protein accumulates to higher levels in individual cancer cells. Geminin is similarly over-expressed in the majority of cancer cell lines tested. The relative ratios of Cdt1 and geminin differ significantly amongst cell lines. Our data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells.