Loss of cell polarity protein Lgl2 in foveolar-type gastric dysplasia: correlation with expression of the apical marker aPKC-zeta

Recognition of gastric epithelial dysplasia, although a key to cancer prevention, can be challenging. In this study, we evaluated whether Lgl2 can serve as a marker of gastric foveolar-type dysplasia. Since atypical protein kinase C (aPKC) is a partner of Lgl2 in the control of apical–basal polarity we also investigated whether aPKC-zeta can compliment Lgl2 as a marker of dysplasia. Routinely processed specimens included 64 normal mucosa, 35 reactive gastropathies, 31 chronic gastritides, 65 gastric dysplasias (25 foveolar; 40 adenomatous), and 34 gastric adenocarcinomas. Twenty (80%) foveolar-type dysplasias showed absence of Lgl2 immunoreactivity, while normal basolateral expression of Lgl2 was consistently seen in normal gastric epithelium (n = 20) and chronic gastritis (n = 22; p < 0.00001). Loss of Lgl2 was similar in the groups with low-grade and high-grade foveolar-type dysplasia, 79% and 83%, respectively. Linear apical anti-aPKC-zeta immunoreactivity was consistently present in the normal epithelium and was preserved in 91% of reactive gastropathies and 87% of chronic gastritides. In contrast, loss of apical aPKC-zeta staining was observed in 47% and 65% of low-grade dysplasias of foveolar and adenomatous types, respectively (p < 0.005) and in nonsignificantly higher percentage of high-grade dysplasias. Apical aPKC-zeta staining was lost in 97% of gastric adenocarcinomas. Our data suggest a role of Lgl2 immunohistochemistry as an adjunct in the diagnosis of foveolar-type gastric dysplasia. aPKC-zeta had moderate sensitivity as a marker of gastric dysplasia and additional studies are needed to establish its role in the diagnosis of dysplasia.     

Mutations in the planar cell polarity genes CELSR1 and SCRIB are associated with the severe neural tube defect craniorachischisis

Craniorachischisis (CRN) is a severe neural tube defect (NTD) resulting from failure to initiate closure, leaving the hindbrain and spinal neural tube entirely open. Clues to the genetic basis of this condition come from several mouse models, which harbor mutations in core members of the planar cell polarity (PCP) signaling pathway. Previous studies of humans with CRN failed to identify mutations in the core PCP genes, VANGL1 and VANGL2. Here, we analyzed other key PCP genes: CELSR1, PRICKLE1, PTK7, and SCRIB, with the finding of eight potentially causative mutations in both CELSR1 and SCRIB. Functional effects of these unique or rare human variants were evaluated using known protein-protein interactions as well as subcellular protein localization. While protein interactions were not affected, variants from five of the 36 patients exhibited a profound alteration in subcellular protein localization, with diminution or abolition of trafficking to the plasma membrane. Comparable effects were seen in the crash and spin cycle mouse Celsr1 mutants, and the line-90 mouse Scrib mutant. We conclude that missense variants in CELSR1 and SCRIB may represent a cause of CRN in humans, as in mice, with defective PCP protein trafficking to the plasma membrane a likely pathogenic mechanism.     

Sequential changes in cellular properties accompanying amniote somite formation

Somites are transient structures derived from the pre‐somitic mesoderm (PSM), involving mesenchyme‐to‐epithelial transition (MET) where the cells change their shape and polarize. Using Scanning electron microscopy (SEM), immunocytochemistry and confocal microscopy, we study the progression of these events along the tail‐to‐head axis of the embryo, which mirrors the progression of somitogenesis (younger cells located more caudally). SEM revealed that PSM epithelialization is a gradual process, which begins much earlier than previously thought, starting with the dorsalmost cells, then the medial ones, and then, simultaneously, the ventral and lateral cells, before a somite fully separates from the PSM. The core (internal) cells of the PSM and somites never epithelialize, which suggests that the core cells could be ‘trapped’ within the somitocoele after cells at the surfaces of the PSM undergo MET. Three‐dimensional imaging of the distribution of the cell polarity markers PKCζ, PAR3, ZO1, the Golgi marker GM130 and the apical marker N‐cadherin reveal that the pattern of polarization is distinctive for each marker and for each surface of the PSM, but the order of these events is not the same as the progression of cell elongation. These observations challenge some assumptions underlying existing models of somite formation.     

Adhesion of Epstein-Barr virus-positive natural killer cell lines to cultured endothelial cells stimulated with inflammatory cytokines

Chronic active Epstein-Barr virus (EBV) infection (CAEBV) is characterized by chronic recurrent infectious mononucleosis-like symptoms. Approximately one-fourth of CAEBV patients develop vascular lesions with infiltration of EBV-positive lymphoid cells. Furthermore, EBV-positive natural killer (NK)/T cell lymphomas often exhibit angiocentric or angiodestructive lesions. These suggest an affinity of EBV-positive NK/T cells to vascular components. In this study, we evaluated the expression of adhesion molecules and cytokines in EBV-positive NK lymphoma cell lines, SNK1 and SNK6, and examined the role of cytokines in the interaction between NK cell lines and endothelial cells. SNKs expressed intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) at much higher levels than those in EBV-negative T cell lines. SNKs produced the larger amount of tumour necrosis factor (TNF)-alpha, which caused increased expression of ICAM-1 and VCAM-1 in cultured human endothelial cells, than that from EBV-negative T cell lines. Furthermore, SNKs exhibited increased adhesion to cultured endothelial cells stimulated with TNF-alpha or interleukin (IL)-1beta, and the pretreatment of cytokine-stimulated endothelial cells with anti-VCAM-1-antibodies reduced cell adhesion. These indicate that the up-regulated expression of VCAM-1 on cytokine-stimulated endothelial cells would be important for the adhesion of EBV-positive NK cells and might initiate the vascular lesions.     

Loss of endothelial planar cell polarity and cellular clearance mechanisms in age-related macular degeneration

Apoptosis, autophagosomes, and lysosomes are lacking in the retinal pigment epithelium (RPE) of age-related macular degeneration (AMD) eyes. Necrosis, not apoptosis, appeared to be the prominent type of cell death in RPE, which led to the accumulation of cell debris within and on both sides of Bruch’s membrane. The endothelium of the choriocapillaris had an altered planar cell polarity which encompassed the disappearance of fenestrations, the thickening of cytoplasm, and anterior nuclear dislocation. There were no significant differences in RPE and choroidal aberrations between macular and temporal regions. Loss of endothelial polarity could be at the crux of AMD initiation and progression.     

Isoform-specific interaction of Flamingo/Starry Night with excess Bazooka affects planar cell polarity in the Drosophila wing.

Epithelia display two types of polarity, apical-basal and planar cell polarity (PCP), and both are crucial for morphogenesis and organogenesis. PCP signaling pathways comprise transmembrane proteins, such as Flamingo/Starry Night, and cytoplasmic, membrane-associated proteins such as Dishevelled. During establishment of PCP in the Drosophila wing, PCP proteins accumulate apically in distinct "cortical domains" on proximal and distal plasma membranes. This finding suggests that their localized function depends on prior definition of apicobasal polarity. Here, we show that overexpression of Bazooka, a PDZ-domain protein essential for apicobasal polarity in the embryo, perturbs development of PCP, but has no effect on apicobasal polarity. The PCP phenotype is associated with a failure to restrict Flamingo/Starry night to the proximal and distal plasma membranes of the wing epithelium. We further demonstrate that flamingo expresses two differentially spliced RNAs in wing imaginal discs, which encode two isoforms of the atypical cadherin Flamingo. The predominant Starry night-type form contains a PDZ-binding motif, which mediates binding to Bazooka in vitro. Pull-down assays support the occurrence of such an interaction in wing imaginal discs. The results suggest that interaction between the apicobasal and planar cell polarity systems has to be tightly coordinated to ensure proper morphogenesis of the wing disc epithelium.     

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.     

Identification and characterization of novel rare mutations in the planar cell polarity gene PRICKLE1 in human neural tube defects

The planar cell polarity (PCP) pathway controls the process of convergent extension (CE) during gastrulation and neural tube closure, and has been implicated in the pathogenesis of neural tube defects (NTDs) in animal models and human cohorts. In this study, we analyzed the role of one core PCP gene PRICKLE1 in these malformations. We screened this gene in 810 unrelated NTD patients and identified seven rare missense heterozygous mutations that were absent in all controls analyzed and predicted to be functionally deleterious using bioinformatics. Functional validation of five PRICKLE1 variants in a zebrafish model demonstrated that one variant, p.Arg682Cys, antagonized the CE phenotype induced by the wild-type zebrafish prickle1a (zpk1a) in a dominant fashion. Our study demonstrates that PRICKLE1 could act as a predisposing factor to human NTDs and further expands our knowledge of the role of PCP genes in the pathogenesis of these malformations.     

Attenuation of experimental arthritis in TRPV1R knockout mice

The Transient Receptor Potential Vanilloid 1 (TRPV1R) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. TRPV1Rs respond to a variety of noxious stimuli including capsaicin, intense heat and acid. These factors, combined with behavioral studies, show that TRPV1Rs are involved in nociception. The aim of our study was to determine whether TRPV1Rs play a role in the development and maintenance of inflammation and mechanical hyperalgesia by studying the development of unilateral joint inflammation in TRPV1R-/- mice. Knee joints of TRPV1R-/- or wild-type (WT) mice were injected with FCA (200 microg) under temporary anesthesia, and the resulting inflammation and hyperalgesia measured for 35 days. Histological analysis was performed on joints at the end of the study. TRPV1R-/- mice developed mild joint swelling which was significantly less than that obtained in WT mice (P < 0.05, Mann-Whitney). The ratio of the weight distribution between the hind limbs in TRPV1R-/- mice was also significantly less than in WT mice (P < 0.05, Mann-Whitney). Neither swelling nor hypersensitivity was completely absent in the knockout mice, indicating either that other mechanisms are involved or that a compensatory mechanism operates in TRPV1R-/- mice. These results suggest that TRPV1 receptors are important for the development of joint inflammation and the associated mechanical hypersensitivity observed in this model.     

Mapping cellular processes in the mesenchyme during palatal development in the absence of Tbx1 reveals complex proliferation changes and perturbed cell packing and polarity

The 22q11 deletion syndromes represent a spectrum of overlapping conditions including cardiac defects and craniofacial malformations. Amongst the craniofacial anomalies that are seen, cleft of the secondary palate is a common feature. Haploinsufficiency of TBX1 is believed to be a major contributor toward many of the developmental structural anomalies that occur in these syndromes, and targeted deletion of Tbx1 in the mouse reproduces many of these malformations, including cleft palate. However, the cellular basis of this defect is only poorly understood. Here, palatal development in the absence of Tbx1 has been analysed, focusing on cellular properties within the whole mesenchymal volume of the palatal shelves. Novel image analyses and data presentation tools were applied to quantify cell proliferation rates, including regions of elevated as well as reduced proliferation, and cell packing in the mesenchyme. Also, cell orientations (nucleus–Golgi axis) were mapped as a potential marker of directional cell movement. Proliferation differed only subtly between wild‐type and mutant until embryonic day (E)15.5 when proliferation in the mutant was significantly lower. Tbx1^?/? palatal shelves had slightly different cell packing than wild‐type, somewhat lower before elevation and higher at E15.5 when the wild‐type palate has elevated and fused. Cell orientation is biased towards the shelf distal edge in the mid‐palate of wild‐type embryos but is essentially random in the Tbx1^?/? mutant shelves, suggesting that polarised processes such as directed cell rearrangement might be causal for the cleft phenotype. The implications of these findings in the context of further understanding Tbx1 function during palatogenesis and of these methods for the more general analysis of genotype–phenotype functional relationships are discussed.     

Loss of nectin‐3 expression as a marker of tumor aggressiveness in pancreatic neuroendocrine tumor

Pancreatic neuroendocrine tumors (PanNETs) are rare, and prediction of aggressive characteristics, such as recurrence and metastasis and prognosis of PanNETs remain difficult. Nectins are cell adhesion molecules that regulate the formation of adherens and tight junctions. In this study, we investigated the clinicopathological significance of nectin‐3 expression in patients with PanNETs. Immunohistochemical analysis of nectin‐3 expression was performed on 78 cases of PanNET. Low nectin‐3 expression in the membrane (positive ratio ≤25%) was observed in 62 cases (79.5%) and was significantly correlated with larger tumor size (>20 mm; P = 0.003), G2/G3 tumors (P = 0.025), higher Ki67 labeling index (≥3%; P = 0.009), lymphatic involvement (P = 0.047), advanced pT‐factor (T2–T4; P = 0.003), lymph node metastasis (P = 0.006), advanced Union for International Cancer Control/American Joint Committee on Cancer‐stage (Stage II–IV; P = 0.001), advanced ENETS stage (Stage IIa–IV; P = 0.001), nonfunctioning tumors (P = 0.002), and a shorter disease‐free survival (P = 0.019). However, there was no significant correlation between nectin‐3 expression in the membrane and/or cytoplasm and the clinicopathological parameters. The present results suggest that decreased nectin‐3 expression in the membrane is associated with increased tumor aggressiveness of PanNETs. Clinically, immunohistochemical analysis of nectin‐3 may help predict tumor aggressiveness for PanNETs.     

Cerebellar allografts in brain of Quaking mice

Summary Fragments of normal embryonic cerebellum were transplanted into adult Quaking mice to examine using peroxidase-antiperoxidase immunocytochemistry the development of genetically normal tissue in an abnormal host environment. The Quaking mouse animals used as hosts are characterized by defective myelin associated glycoprotein. Normal characteristic expression and distribution of neurofilaments was observed in the cerebellar grafts. Nonphosphorylated epitopes of neurofilaments were seen in Purkinje cell bodies and dendrites. The phosphorylated epitopes of neurofilaments were observed in basket cell axons. Phosphorylated epitopes were also present in numerous myelinated axons, which were probably fibers from deep cerebellar nuclei. These data support the notion that neurogenesis with alternative connections can occur in transplants. Staining patterns with myelin associated glycoprotein and myelin basic protein also suggest that normal myelination occurs in grafts transplanted to the brains of Quaking mice.     

Anatomically discrete sex differences and enhancement by testosterone of cell proliferation in the telencephalic ventricle zone of the adult canary brain

Previous work in songbirds has suggested that testosterone increases neuronal recruitment and survival in HVC but does not affect neuronal proliferation in the ventricular zone and that males and females have similar rates of proliferation except at discrete locations. Many of these conclusions are however based on limited data or were inferred indirectly. Here we specifically tested the effects of testosterone on cellular proliferation in the ventricular zone of both male and female adult canaries. We implanted adult birds of both sexes with testosterone or empty implants for 1 week and injected them with BrdU. One day later, we collected their brains and quantified BrdU-positive cells in the ventricular zone (VZ) at different rostro-caudal levels of the brain, ranging from the level where the song nucleus Area X occurs through the caudal extent of HVC. Proliferation in the dorsal part of the VZ was low and unaffected by sex or testosterone treatment. In the ventral part of the VZ, females had more proliferating cells than males, but only at rostral levels, near Area X. Also in the ventral part of the VZ, testosterone increased proliferation in birds of both sexes, but only in the mid- to caudal-VZ, caudal to the level of Area X, around the septum and HVC. We thus demonstrate here that there is both an effect of testosterone and possibly a more subtle effect of sex on cellular proliferation in the adult songbird brain, and that these effects are specific to different levels of the brain.     

The Pim-1 kinase stimulates maturation of TCRbeta-deficient T cell progenitors: implications for the mechanism of Pim-1 action

We demonstrate that overexpression of Pim-1, a cytoplasmic serine/threonine kinase of poorly defined function, results in the development of substantial numbers of CD4(+)CD8(+) double-positive thymocytes in two independent knock-out mouse models (i.e. the RAG-1-deficient and TCRbeta gene enhancer-deleted mice) in which production of a functionally rearranged TCRbeta gene (hence the pre-TCR) is impaired. This activity of Pim-1, however, does not affect signaling through the Ras/Raf/MAP kinase cascade nor signaling which mediates suppression of TCRbeta gene recombination (i.e. allelic exclusion). While overexpression of Pim-1 positively affects cell cycle progression in selected CD4(-)CD8(-) double-negative precursors, it did not affect expression of components of the cell cycle machinery, with the exception of the G(1)-specific phosphatase Cdc25A upon antigen receptor stimulation. We propose that Pim-1 acts downstream, or in parallel, to pre-TCR-mediated selection as one factor involved in the proliferative expansion of beta-selected pre-T cells.     

Transplantation of a human ovarian cystadenocarcinoma into severe combined immunodeficient (SCID) mice — formation of metastases without significant alteration of the tumour cell phenotype

Human ovarian papillary cystadenocarcinoma cells were injected intraperitoneally into severe combined immunodeficient (SCID) mice. After intraperitoneal application the cells, designated SoTü, grew well in vivo , lodged on to the peritoneum, formed local metastatic deposits, led to the development of ascites in the mice and formed distant metastases in the lungs. If lodged in the ovary, the morphology of the SoTu¨ tumour remarkably resembled that of the primary tumour in the patient. In contrast, several attempts failed to maintain the SoTü cells in vitro . If SCID mouse ascites derived SoTü were transplanted subcutaneously in SCID mice, they formed cystic tumours which also metastasized into the lungs. Immunophenotypical analysis of cell adhesion molecule expression, cell proliferation markers, various oncoproteins, keratin, vimentin, and lectin binding site expression all showed striking similarity between the primary tumour and the SCID mouse explants. In particular, expression of binding sites for the lectin Helix pomatia agglutinin (HPA), which has been shown to be an index of metastatic potential in several human carcinomas, was found on the primary tumour as well as on tumour cells grown in SCID mice, indicating that HPA might be a prognostic indicator in ovarian carcinoma as well. Our results demonstrate that the human/SCID mouse system can mimic growth and distant metastasis formation of human ovarian carcinoma. Although the formation of distant metastases is a relatively rare event in patients, this model system might help to elucidate mechanisms of metastasis formation in ovarian cancer.     

Loss of dystrophin and the microtubule‐binding protein ELP‐1 causes progressive paralysis and death of adult C. elegans

EMAP‐like proteins (ELPs) are conserved microtubule‐binding proteins that function during cell division and in the behavior of post‐mitotic cells. In Caenorhabditis elegans, ELP‐1 is broadly expressed in many cells and tissues including the touch receptor neurons and body wall muscle. Within muscle, ELP‐1 is associated with a microtubule network that is closely opposed to the integrin‐based adhesion sites called dense bodies. To examine ELP‐1 function, we utilized an elp‐1 RNA interference assay and screened for synthetic interactions with mutated adhesion site proteins. We reveal a synthetic lethal relationship between ELP‐1 and the dystrophin‐like protein, DYS‐1. Reduction of ELP‐1 in a dystrophin [dys‐1(cx18)] mutant results in adult animals with motility defects, splayed and hypercontracted muscle with altered cholinergic signaling. Worms fill with vesicles, become flaccid, and die. We conclude that ELP‐1 is a genetic modifier of a C. elegans model of muscular dystrophy. Developmental Dynamics, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of an imprinting procedure on cell proliferation in the chick brain

We report here studies on the effects of an imprinting procedure on cell proliferation in neonatal chicks in brain structures known to undergo plastic changes in imprinting. Proliferating cells were detected immunohistochemically on brain sections by incorporation of pre-training doses of 5-bromodeoxyuridine (BrdU) into DNA; numbers of new cells were counted in the intermediate medial mesopallium, the intermediate arcopallium, the medial part of the mesopallium and the nidopallium, the dorsocaudal nidopallium, the hippocampus, and the parahippocampal region 24 h and seven days after training. The intermediate medial mesopallium showed an increase in the number of BrdU-positive cells 24 h after training. However, at seven days post-training, the number of BrdU-containing cells decreased in the medial nidopallium and mesopallium, in the dorsocaudal nidopallium, and the right intermediate medial mesopallium. Thus, the imprinting procedure had differently directed transient and long-term influences on the genesis of new cells in the chick brain, inducing the appearance of a large number of cells in the parenchyma of the brain one day after training and decreases in the numbers of cells at later time points. This double effect may be associated with the fact that the imprinting procedure simultaneously initiates two brain processes involving the control of cell proliferation — one related to maturation of a species-specific functional system for tracking individuals of the same species and one related to remembering the characteristics of the actual parent. __________ Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti imeni I. P. Pavlova, Vol. 57, No. 2, pp. 181–190, March–April, 2007.     

Detection of nucleosome–IgG immune complexes in ascites from mice transplanted with anti-DNA antibody-secreting hybridomas and in plasmas from MRL-lpr/lpr mice

Autoantibodies directed against chromatin components characterize lupus diseases. Immune complexes made of these autoantibodies bound to nucleosomes released from dead cells could play some pathogenic role. The aims of this study were to investigate if nucleosome–IgG complexes could contaminate IgG anti-DNA MoAb preparations, and if such complexes circulate in lupus diseases. A new method was set up using preformed nucleosome–IgG complexes. Complexes were adsorbed onto microplate through Fc binding and nucleosomal DNA was detected by internal incorporation of labelled nucleotide. Using this method, high amounts of complexes were found in ascites from mice transplanted with anti-DNA antibody-secreting hybridomas. In some ascites, nucleosome was found to be strongly associated with the MoAb, confirming that nucleosome–IgG complexes could contaminate monoclonal autoantibody preparations. In MRL-lpr/lpr mice, nucleosome–IgG complexes were detected at 16–24 weeks of age at a time when kidney lesions are rapidly worsening, raising the question of their pathogenic significance.