Licensing of DNA replication, cancer, pluripotency and differentiation: an interlinked world?

Recent findings provide evidence for a functional interplay between DNA replication and the seemingly distinct areas of cancer, development and pluripotency. Protein complexes participating in DNA replication origin licensing are now known to have roles in development, while their deregulation can lead to cancer. Moreover, transcription factors implicated in the maintenance of or reversal to the pluripotent state have links to the pre-replicative machinery. Several studies have shown that overexpression of these factors is associated to cancer.

Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing.

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a "permissive" heterotrimer and an "inhibitory" heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.

Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle.

Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.

Alpha 2-adrenergic receptors decrease DNA replication and cell proliferation and induce neurite outgrowth in transfected rat pheochromocytoma cells.

Alpha 2-adrenergic receptors (alpha(2)-ARs) have a widespread distribution in the central nervous system (CNS) and affect a number of biochemical and behavioral functions, including stimulation of prefrontal cortex (PFC) and cognitive function. In addition to its role as a classical neurotransmitter, norepinephrine (NE) has been recently shown to exert an important influence on the plasticity in areas of the brain where neurogenesis persists in the adult, notably the subgranular zone (SGZ) within the dentate gyrus of the hippocampus and the olfactory bulb (OB). In regulating adult neurogenesis, the noradrenergic system is functionally integrated with chronic stress and depression. Chronic stress, depression, or depletion of NE in vivo suppress, and antidepressant treatments induce hippocampal neurogenesis by down- or upregulating, respectively, cell proliferation. In the present study we show that alpha(2)-AR subtypes promote the differentiation rather than cell proliferation of PC12 cells. It is conceivable that alpha(2)-ARs might contribute neurotrophic actions in vivo synergistically or in permutation with other neurotrophic factors.

ILK over-expression in human colon cancer progression correlates with activation of beta-catenin, down-regulation of E-cadherin and activation of the Akt-FKHR pathway.

Integrin-linked kinase (ILK) has been implicated in the development and progression of several human malignancies. However, the role of ILK in human colon cancer progression is not well established, neither have its possible in vivo downstream effectors in the disease been identified. We studied, by immunohistochemistry, ILK, beta-catenin, E-cadherin, p-Akt and p-FKHR protein expression in 125 primary colon carcinomas and 45 corresponding lymph node metastases. ILK was expressed in 98.4% of the primary tumours and in 100% of metastatic lesions. The levels of ILK expression correlated strongly with tumour invasion, tumour grade and stage and were significantly higher in metastatic tumours. Activation of beta-catenin, down-regulation of E-cadherin and activation of the Akt-FKHR pathway correlated significantly with both ILK expression and tumour progression parameters. In conclusion, our results suggest that ILK may have an important role in progression of human colon cancer, possibly through in vivo regulation of beta-catenin, E-cadherin and Akt pathways. Our study also provides some evidence implicating p-FKHR in human colon carcinogenesis and ILK signalling.

Expression of the licensing factors, Cdt1 and Geminin, in human colon cancer.

Licensing of chromatin for replication is an evolu-tionarily conserved step in the control of cell division and genomic integrity. Proteins that participate in licensing have been recently documented to denote the proliferative state of cells and they have been proposed as diagnostic and prognostic markers in human cancer. Cdt1 was recently discovered as an important licensing factor, that is inhibited by Geminin. In the present study we analyzed Cdt1 and Geminin expression in human colon cancer. We showed that Cdt1 protein is highly expressed in human neoplastic lesions of the colon while its cell-cycle phase-specific expression profile appears preserved during human carcinogenesis. Similarly, Geminin, Cdt1's inhibitor, is also overexpressed in colon carcinomas and its expression correlates with significant clinicopathological parameters of the disease. Moreover, both Cdt1 and Geminin expression are severely downregulated upon differentiation of Caco-2 cells, an in vitro model of intestinal epithelial differentiation.

The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase.

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.

Identification of groupings of graph theoretical molecular descriptors using a hybrid cluster analysis approach.

There is an abundance of structural molecular descriptors of various forms that have been proposed and tested over the years. Very often different descriptors represent, more or less, the same aspects of molecular structures and, thus, they have diminished discriminating power for the identification of different structural features that might contribute to the molecular property, or activity of interest. Therefore, it is essential that noncorrelated descriptors be employed to ensure the wider and the less inflated possible coverage of the chemical space. The most usual approach for reducing the number of descriptors and employing noncorrelated (or orthogonal) descriptors involves principal component analysis (PCA) or other factor analytical techniques. In this work we present an approach for determining relationships (groupings) among 240 graph-theoretical descriptors, as a means for selecting nonredundant ones, based on the application of cluster analysis (CA). To remove inherent biases and particularities of different CA algorithms, several clustering solutions, using these algorithms, were "hybridized" to obtain a reliable and confident overall solution concerning how the interrelationships within the data are structured. The calculated correlation coefficients between descriptors were used as a reference for a discussion on the different CA methods employed, and the resulted clusters of descriptors were statistically analyzed for deriving the intercorrelations between the different operators, weighting schemes and matrices used for the computation of these descriptors.

Primary structure, chromosomal mapping, expression and transcriptional activity of murine hepatocyte nuclear factor 4gamma.

We demonstrate the presence of a new member of the orphan nuclear receptor hepatocyte nuclear factor 4 (HNF4) subfamily in mouse which is genetically distinct from the previously characterized mouse HNF4alpha gene. The new member of the HNF4 subfamily shows highest amino acid identity, similar tissue distribution and syntenous chromosomal localization to the recently described human HNF4gamma (NR2A2), we therefore classify it as mouse HNF4gamma (mHNF4gamma). A combination of RT-PCR and immunohistochemical analysis showed expression of mHNF4gamma mRNA and protein in the endocrine pancreas, testes, kidney and gut. By co-transfection experiments, we show that mHNF4gamma is able to activate transcription, acting through binding sites that have been previously characterized as HNF4alpha binding sites. The presence of HNFgamma in human and mouse implies that a complex transcriptional network exists in higher vertebrates involving a number of HNF4 members with overlapping yet distinct function and tissue distribution.

Development of the mammalian enteric nervous system.

The mammalian enteric nervous system is derived from neural crest cells which invade the foregut and hindgut mesenchyme. It has been established that signalling molecules produced by the mesenchyme of the gut wall play a critical role in the development of the mammalian enteric nervous system. Recent studies have characterised further the role of such molecules and have identified novel extracellular and intracellular signals that are critical for enteric ganglia formation.

Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system.

RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.

III. Role Of the RET signal transduction pathway in development of the mammalian enteric nervous system.

The enteric nervous system (ENS) in vertebrates is derived from the neural crest and constitutes the most complex part of the peripheral nervous system. Natural and induced mutagenesis in mammals has shown that the tyrosine kinase receptor RET and its functional ligand glial cell line-derived neurotrophic factor (GDNF) play key roles in the development of the ENS in humans and mice. We have developed and briefly describe here a number of assays that analyze the specific function of the RET receptor and its ligand. Our data suggest that the RET signal transduction pathway has multiple roles in the development of the mammalian ENS.

Generation of inhibitory mutants of hepatocyte nuclear factor 4.

Hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear-receptor gene superfamily. HNF-4 binds to response elements of several liver-enriched genes and exhibits a restricted pattern of expression, suggesting an important role for HNF-4 in tissue-specific gene regulation. Here, we report the generation of three mutated forms of the HNF-4 protein, their effects on the ability of the protein to transactivate through HNF-4-response elements, and their ability to suppress transactivation by the wild-type protein. Two mutated forms of the HNF-4 protein, one in which the DNA-binding domain has been deleted and another in which the HNF-4 proximal box has been replaced by that of the glucocorticoid receptor, behaved as inhibitors of the wild-type protein. The properties of a carboxy-terminal-deletion mutant allow us to propose a region of HNF-4 involved in transactivation.

Characterization of the mouse HNF-4 gene and its expression during mouse embryogenesis.

Hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear receptor gene superfamily with unknown ligand. It has been assumed to play an important role in the regulation of gene expression in the liver. Here, we report the cloning and characterization of the mouse HNF-4 gene, as well as its expression during embryogenesis. The HNF-4 protein is encoded by ten exons. The gene structure is unique in the steroid receptor superfamily in that the second zinc finger is encoded by two exons. HNF-4 mRNA is expressed in a limited number of mouse adult tissues: liver, kidney, intestine, stomach and skin. HNF-4 could play an important role in the formation and function of visceral yolk sac and in the development of the liver and kidney since its mRNA, as determined by in situ hybridization, appears upon primary differentiation of these organs. As a first step in the study of the regulatory elements of the HNF-4 gene, we mapped the transcription start site and carried out DNase I hypersensitive site (HS) analysis over a region of approximately 22kb upstream of the gene. The complexity of the HSs suggests that multiple elements might contribute to the transcriptional regulation of the HNF-4 gene.