Phosphorylation of NHERF1 S279 and S301 differentially regulates breast cancer cell phenotype and metastatic organotropism

Metastatic cancer cells are highly plastic for the expression of different tumor phenotype hallmarks and organotropism. This plasticity is highly regulated but the dynamics of the signaling processes orchestrating the shift from one cell phenotype and metastatic organ pattern to another are still largely unknown. The scaffolding protein NHERF1 has been shown to regulate the expression of different neoplastic phenotypes through its PDZ domains, which forms the mechanistic basis for metastatic organotropism. This reprogramming activity was postulated to be dependent on its differential phosphorylation patterns. Here, we show that NHERF1 phosphorylation on S279/S301 dictates several tumor phenotypes such as in vivo invasion, NHE1-mediated matrix digestion, growth and vasculogenic mimicry. Remarkably, injecting mice with cells having differential NHERF1 expression and phosphorylation drove a shift from the predominantly lung colonization (WT NHERF1) to predominately bone colonization (double S279A/S301A mutant), indicating that NHERF1 phosphorylation also acts as a signaling switch in metastatic organotropism.     

The basolateral sorting signals of the thyrotropin and luteinizing hormone receptors: an unusual family of signals sharing an unusual distal intracellular localization, but unrelated in their structures

The mechanisms of the basolateral targeting of G protein-coupled receptors remain largely unknown. Mutagenesis experiments have allowed us to identify the basolateral sorting signals of the TSH and LH receptors expressed in Madin-Darby canine kidney cells and thyroid follicular FRT cells. Unexpectedly these signals (amino acids 731-746 and 672-689, respectively) share an unusual localization in the distal part of the intracellular domain of the receptors at a marked distance from the membrane. When grafted onto the p75-neurotropin receptor, these signals redirect this normally apically expressed protein to the basolateral cell surface. They are independent of the endocytosis signal. The basolateral sorting signals of TSH, LH, and FSH receptors do not exhibit primary sequence homology with each other or with any other known signal. Furthermore, circular dichroism studies show that the three signals exhibit distinct secondary structures. The TSH receptor has a stable helical structure, the LH receptor has both helix and beta-sheet structures, and the FSH receptor sorting signal has a main random coil structure. This means that even in closely-related receptors different secondary structures can be found for basolateral signals unrelated to internalization signals. This observation contrasts with what is known about basolateral signals related to internalization signals for which a common beta-turn structure has been described. Deletion of the basolateral sorting signals results in apical targeting of the receptors, suggesting the existence of apical sorting information. However, a soluble form of the TSH receptor, which harbors all N- and putative O-linked oligosaccharides, is secreted in a nonpolarized fashion. This implies that apical sorting information must be located elsewhere, either in the transmembrane or in the intracellular domains of the receptor.     

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.     

Report from the in vitro micronucleus assay working group

At the Washington "2nd International Workshop on Genotoxicity Testing" (25-26 March 1999) current methodologies and data for the in vitro micronucleus test were reviewed. As a result, guidelines for the conduct of specific aspects of the protocol were developed. Agreement was achieved on the following topics: choice of cells, slide preparation, analysis of micronuclei, toxicity, use of cytochalasin-B, number of doses, and treatment/harvest times [Environ. Mol. Mutagen. 35 (2000) 167]. Because there were a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at that time. These studies have now been completed and the data were reviewed at the Plymouth "3rd International Workshop on Genotoxicity Testing" (28-29 June 2002). Data from studies coordinated by the French Society of Genetic Toxicology, Japanese collaborative studies, European pharmaceutical industry validation studies, along with data from Lilly Research Laboratories were used to prepare conclusions on the main aspects of the in vitro micronucleus protocol. In this paper, the consensus agreements on the protocol for performing the in vitro micronucleus assay are presented. The major recommendations concern: 1. Demonstration of cell proliferation: both cell lines and lymphocytes can be used, but demonstration of cell proliferation in both control and treated cells is compulsory for the acceptance of the test. 2. Assessment of toxicity and dose range finding: assessment of toxicity should be performed by determining cell proliferation, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; and by determining other markers for cytotoxicity (confluency, apoptosis, necrosis) which can provide valuable additional information. 3. Treatment schedules for cell lines and lymphocytes. 4. Choice of positive controls: without S9-mix both a clastogen (e.g. mitomycin C or bleomycin) and an aneugen (e.g. colchicine) should be included as positive controls and a clastogen that requires S9 for activity when S9-mix is used (e.g. dimethylnitrosamine, or cyclophosphamide in those cell types that cannot activate this agent directly). 5. Duplicate cultures and number of cells to be scored. 6. Repeat experiments: in lymphocytes, for each experiment blood from 2 different healthy young and non-smoking donors should be compared. In cell lines, the experiments need only to be repeated if the first one is negative. 7. Statistics: statistical significance should not be the sole factor for determining positive results. Biological meaning should serve as a guideline. Examples of statistical analyses are given.     

Importance of detecting numerical versus structural chromosome aberrations

The aim is to review briefly the key questions related to aneuploidy/polyploidy and to compare the advantages and disadvantages of the in vitro micronucleus test to assess aneuploidy/polyploidy in vitro. The key questions that will be addressed, concern the importance of polyploidy for health, and cancer in particular, the mechanisms leading to aneuploidy and polyploidy, and the survival of aneuploid/polyploid cells.The recently recognised contribution of numerical chromosome changes to carcinogenesis triggered the development and the implementation of tests specifically aiming at the detection of aneugens in the test battery for mutagenicity and carcinogenicity. The validation of the in vitro micronucleus test in combination with the identification of in vitro divided cells with the cytokinesis-block methodology and of centromeres with pancentromeric or chromosome specific centromeric probes fluorescence in situ hybridisation (FISH) provides a sensitive, easy to score and powerful test which allows assessment of cell proliferation, the discrimination between chromosome breaks, chromosome loss and chromosome non-disjunction and polyploidy. Moreover, classic histology permits the estimation of necrosis and apoptosis on the same slide. The cytokinesis-blocked micronucleus assay could be considered as a multi-endpoint test for genotoxic responses to clastogens/aneugens. This methodology has also shown to be capable of identifying threshold values for the induction of chromosome loss and/or non-disjunction by microtubule inhibitors, data which are particularly important for risk calculations. Similar approaches were conducted in vivo on bone marrow in mice and rats (except for identification of chromosome non-disjunction), and are in development for gut in mice.     

LPS-induced lung injury in neonatal rats: changes in gelatinase activities and consequences on lung growth

Postnatal lung growth disorders may involve imbalance between metalloproteinases and their inhibitors. Inflammatory cell 92-kDa gelatinase overactivity has been reported in adults with lung injury but has not been looked for in neonates. We compared gelatinase activity in neonatal and adult rats and evaluated postnatal lung growth after lipopolysaccharide (LPS)-induced lung injury. Significant intra-alveolar inflammatory cell recruitment occurred in adults and neonates; cell counts increased 16-fold in adults and 2.7-fold in neonates. Total 92-kDa gelatinase activity was increased in neonates and adults and was significantly correlated to inflammatory cell counts. For a given cell count, 92-kDa gelatinase increased more in neonates than in adults. Morphometric neonatal lung analysis showed that LPS-injured lungs had decreases in absolute values of lung volume (P < 0.03), alveolar surface (P < 0.004), and air space volume (P < 0.03). Doxycycline, a nonspecific metalloproteinase inhibitor, partly inhibited LPS-induced 92-kDa gelatinase overactivity but did not improve LPS-induced alveolar growth disorders. LPS-mediated lung injury in neonatal rats induced both gelatinase B overactivity and alveolar growth disorders, although no causal link between these two effects was demonstrated.     

The mechanism of the angiotensin-converting enzyme inhibitor quinapril is not related to bradykinin level in heart tissue

In order to examine the effect of the angiotensin-converting enzyme inhibitor (ACEi) quinapril, we performed a sensitive and specific radioimmunoassay (RIA) to quantify bradykinin, BK-(1-9), in heart and kidney tissues. The BK-(1-9) level was unaffected in the heart of sham and water-deprived rats treated for 2h with quinapril (10mg/kg), but was significantly higher in the kidneys in the two groups. In these conditions, circulating and tissue angiotensin II (Ang II) levels were significantly decreased by quinapril. Moreover, our results indicated that acute treatment with this dose of quinapril induced kinin-mediated effects which were not related to its action on bradykinin degradation in rat hearts.     

Sequential protein association with nascent 60S ribosomal particles

Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.     

Discovery of cell-active phenyl-imidazole Pin1 inhibitors by structure-guided fragment evolution

Pin1 is an emerging oncology target strongly implicated in Ras and ErbB2-mediated tumourigenesis. Pin1 isomerizes bonds linking phospho-serine/threonine moieties to proline enabling it to play a key role in proline-directed kinase signalling. Here we report a novel series of Pin1 inhibitors based on a phenyl imidazole acid core that contains sub-μM inhibitors. Compounds have been identified that block prostate cancer cell growth under conditions where Pin1 is essential.     

Integrating multiple analytical approaches to spatially delineate and characterize genetic population structure: an application to boreal caribou (<Emphasis Type="Italic">Rangifer tarandus caribou</Emphasis>) in central Canada

Individual-based clustering (IBC) methods have become increasingly popular for the characterization and delineation of genetic population units for numerous species. These methods delineate populations based on the genetic assumptions of a breeding unit which may provide a better representation of the behaviour of the species. The increasing use of IBC has resulted in the development of several analytical models all of which vary in their theoretical assumptions to infer genetic population structure. In this paper, we report a comparative strategy utilizing three IBC methods to characterize the spatial genetic structure of the boreal population of woodland caribou (Rangifer tarandus caribou) in central Canada. In addition, we implement both tests for isolation-by-distance (IBD) and frequency-based assignment tests to validate the consensus genetic clusters as defined by IBC. We also compare indirect metrics of genetic diversity and gene flow using both a priori defined herds and the IBC defined populations. Although our results show some concordance between both pre-defined herds and IBC derived genetic clusters, the IBC analyses identified a cluster that was cryptic to observation-based caribou herds and found no difference between several adjacent herds. By comparing multiple IBC methods and integrating both IBD and indirect genetic diversity metrics a posteriori, our strategy provides an effective means to delineate wildlife population structure and accurately assess genetic diversity and connectivity.