Gliotypic neural stem cells transiently adopt tumorigenic properties during normal differentiation

An increasing body of evidence suggests that astrocytic gliomas of the central nervous system may be derived from gliotypic neural stem cells. To date, the study of these tumors, particularly the identification of originating cellular population(s), has been frustrated by technical difficulties in accessing the native niche of stem cells. To identify any hallmark signs of cancer in neural stem cells or their progeny, we cultured subventricular zone-derived tissue in a unique in vitro model that temporally and phenotypically recapitulates adult neurogenesis. Contrary to some reports, we found undifferentiated neural stem cells possess few characteristics, suggesting prototumorigenic potential. However, when induced to differentiate, neural stem cells give rise to intermediate progenitors that transiently exhibit multiple glioma characteristics, including aneuploidy, loss of growth-contact inhibition, alterations in cell cycle, and growth factor insensitivity. Further examination of progenitor populations revealed a subset of cells defined by the aberrant expression of (the pathological glioma marker) class III beta-tubulin that exhibit intrinsic parental properties of gliomas, including multilineage differentiation and continued proliferation in the absence of a complex cellular regulatory environment. As tumorigenic characteristics in progenitor cells normally disappear with the generation of mature progeny, this suggests that developmentally intermediate progenitor cells, rather than neural stem cells, may be the origin of so-called "stem cell-derived" tumors.     

A novel 26 kilodalton antigen expressed on the surface membrane of activated T cells

We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) 1B10, raised against an activated gammadelta T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb 1B10 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B10+. 1B10 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) alphabeta+ and gammadelta+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were 1B10+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb 1B10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb 1B10 from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb 1B10 recognizes a novel appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease.     

A cell surface antigen present on cultured cerebellar neurones appears to be transiently expressed during cerebellar development in the rat

A monoclonal antibody has been produced from a fusion of NSO myeloma cells and splenocytes from a mouse immunized with cultures from early postnatal rat cerebellum. The binding of this antibody designated 69A1 is concentrated in the molecular layer of the developing rat cerebellum during the first two weeks postnatally but falls below the level of detection during the third week. Immunoelectron microscopy has shown antibody binding in the molecular layer to be confined to the parallel fibres of the granule neurones. The disappearance of binding coincides with a period during which the formation of new parallel fibres is completed and rapid synaptogenesis within the molecular layer begins.     

Error-prone polyploid mitosis during normal Drosophila development

Endopolyploidy arises during normal development in many species when cells undergo endocycles-variant cell cycles in which DNA replicates but daughter cells do not form. Normally, polyploid cells do not divide mitotically after initiating endocycles; hence, little is known about their mitotic competence. However, polyploid cells are found in many tumors, and the enhanced chromosomal instability of polyploid cells in culture suggests that such cells contribute to tumor aneuploidy. Here, we describe a novel polyploid Drosophila cell type that undergoes normal mitotic cycles as part of a remodeling process that forms the adult rectal papillae. Similar polyploid mitotic divisions, but not depolyploidizing divisions, were observed during adult ileum development in the mosquito Culex pipiens. Extended anaphases, chromosome bridges, and lagging chromosomes were frequent during these polyploid divisions, despite normal expression of cell cycle regulators. Our results show that the switch to endocycles during development is not irreversible, but argue that the polyploid mitotic cycle is inherently error-prone, and that polyploid mitoses may help destabilize the cancer genome.     

Analysis of the tumour-associated antigen TAG-12 by monoclonal antibody 7A9 in normal,benign and malignant mammary tissues

Summary A monoclonal antibody 7A9 was raised against the tumour-associated glycoprotein TAG-12 purified from T47-D breast carcinoma cells. In immunoblots from cytosol of T47-D cells and from sera of breast cancer patients, antibody 7A9 detects the high molecular weight mucin-like TAG-12 antigen. A series of paraffin sections of normal, benign and malignant mammary tissues have been studied with monoclonal antibody 7A9 and the immunoalkaline phosphatase method. In resting gland, proliferating gland and fibroadenoma ducts, reactivity of 7A9 was mainly restricted to luminal membranes of epithelial cells and secretions. 77/79 primary breast carcinomas including ductal, lobular and various other carcinoma types showed cytoplasmic and/or membrane-associated staining with 7A9 in most tumour cells. Metastases (31/31) from different sites were also positive. Strong immunoreactivity with single tumour cells was noted in cytological preparations from freshly resected breast cancer tissue. Thus, monoclonal antibody 7A9 seems to be very useful for the targeting of breast carcinoma cells.     

A novel immunoglobulin superfamily junctional molecule expressed by antigen presenting cells, endothelial cells and platelets.

The architecture of lymphoid microenvironments depends upon complex interactions between several stromal cell types. We describe in this report the cloning of a cDNA which encodes a novel membrane molecule containing two external Ig-like domains. It is expressed at the junction between endothelial cells including HEV. It is also expressed by platelets and MHC class II+ antigen presenting cells in thymic medulla and T-cell areas in peripheral lymphoid organs. These cells which lack in RelB-deficient mice include tissue-derived dendritic, epithelial cells and macrophages. Thus, this molecule might contribute to the organization of cell junctions in different microenvironments.     

An antigen expressed by murine CD8+ T cells and activated B cells

A monoclonal antibody, H1F5, is described that reacts with a subset of Lyt-2 (CD8) mouse T cells and LPS-activated B cells. In both lymph nodes and spleen of BALB/c mice, the H1F5 antigen is coexpressed approximately on 20%-30% of the CD8+ T cells and approximately on 91% of LPS-activated B cells. In the thymus, few cells (less than 1%) are positive for the marker, but no correlation could be demonstrated with markers for mature T cells such as MEL-14 and PNA expression. Elimination of H1F5+ cells by complement lysis led to a 30%-50% reduction of specific lysis as measured in a primary allo CTL, indicating that the cytotoxic effector cells are injured. The relationship of this marker and other antigenic determinants on lymphocytes is discussed.     

A transiently expressed SK current sustains and modulates action potential activity in immature mouse inner hair cells

From just after birth, mouse inner hair cells (IHCs) expressed a Ca²⁺-activated K⁺ current that was reduced by intracellular BAPTA at concentrations ≥ 1 m m . The block of this current by nifedipine suggests the direct involvement of Ca_v1.3 Ca²⁺ channels in its activation. On the basis of its high sensitivity to apamin ( K _D 360 p m ) it was identified as a small-conductance Ca²⁺-activated K⁺ current (SK), probably SK2. A similar current was also found in outer hair cells (OHCs) from the beginning of the second postnatal week. In both cell types the appearance of the SK current coincided with their becoming responsive to acetylcholine (ACh), the main efferent neurotransmitter in the cochlea. The effect of ACh on IHCs was abolished when they were simultaneously superfused with strychnine, consistent with the presence of nicotinic ACh receptors (nAChRs). Extracellular Ca²⁺ either potentiated or blocked the nAChR current depending on its concentration, as previously reported for the recombinant α9α10 nAChR. Outward currents activated by ACh were reduced by blocking the SK current with apamin or by preventing SK current activation with intracellular BAPTA (≥ 10 m m ). The endogenous mobile Ca²⁺ buffer concentration was estimated to be equivalent to about 1 m m BAPTA, suggesting that in physiological conditions the SK channel is significantly activated by Ca²⁺ influx through both Ca_v1.3 Ca²⁺ channels and α9α10 nAChRs. Current clamp experiments showed that in IHCs the SK current is required for sustaining a train of action potentials and also modulates their frequency when activated by ACh.     

Mammalian achaete-scute homolog 1 is transiently expressed by spatially restricted subsets of early neuroepithelial and neural crest cells

Using monoclonal antibodies, we have examined the expression pattern of MASH1, a basic helix-loop-helix protein that is a mammalian homolog of the Drosophila achaete-scute proteins. In Drosophila, achaete-scute genes are required for the determination of a subset of neurons. In the rat embryo, MASH1 expression is confined to subpopulations of neural precursor cells. The induction of MASH1 precedes, but is extinguished upon, overt neuronal differentiation. MASH1 is expressed in the forebrain by spatially restricted domains of neuroepithelium and in the peripheral nervous system exclusively by precursors of sympathetic and enteric neurons. The features of early and transient expression, in spatially restricted subpopulations of neural precursors, are similar to those observed for achaete-scute. Thus, the amino acid sequence conservation between MASH1 and achaete-scute is reflected in a parallel conservation of cell type specificity of expression, similar to the case of mammalian MyoD and Drosophila nautilus. These data support the idea that helix-loop-helix proteins may represent an evolutionarily conserved family of cell-type determination genes, of which MASH1 is the first neural-specific member identified in vertebrates.     

CD40 antigen is expressed by endothelial cells and tumor cells in Kaposi's sarcoma.

The CD40 antigen is a member of the tumor necrosis factor receptor/nerve growth factor receptor superfamily and is involved in cell proliferation, differentiation, and survival. Using different monoclonal antibodies, we found CD40 expression by immunohistochemistry on CD31- and CD34-positive Kaposi's sarcoma spindle cells in all tumors of 18 HIV-1 seropositive and 4 HIV-1 seronegative patients. Western blot analysis of tumor lysates detected a 48- to 50-kd glycoprotein corresponding to the CD40 antigen expressed by B lymphocytes. CD40 expression was also detectable in one of four cultures of spindle cells derived from Kaposi sarcoma tissue. Treatment of the CD40-positive spindle cells but not of the CD40-negative ones with interferon-gamma up-regulated CD40 surface expression. Besides on Kaposi sarcoma tumor cells, CD40 was distinctly present on vascular endothelial cells in areas within and adjacent to the tumors and in benign inflammatory lesions such as granulation tissue of HIV-1-negative patients. In contrast, CD34-negative endothelia of thin walled vessels, most likely lymphatics, were predominantly CD40 negative. Only faint or no CD40 expression was found on endothelial cells in normal skin. We conclude from our data that expression of the CD40 antigen by endothelial cells is up-regulated during tissue inflammation. As signaling through CD40 is able to increase cell survival, expression of CD40 by Kaposi sarcoma tumor cells might play an important role in the pathogenesis of this neoplasm.     

Specificity of antigen recognition by normal thymus cells in nude mice

If injected with normal thymus cells from BALB/c or BALB/c-Ig^b, nude mice from a stock which is partially backcrossed to BALB/c make antibodies to many antigens to which untreated nude mice do not respond. Anti-Ig^b can be made if the thymus donor is BALB/c, but not if the thymus donor is BALB/c-Ig^b. If cells from both donor strains are injected, a response is obtained which shows that the unresponsiveness of recipients of BALB/c-Igb cells is not due to tolerance of donor antigens.     

The control of chromosome segregation during mitosis in epithelial cells by substrate elasticity

Materials of defined elasticity, including synthetic material scaffolds and tissue-derived matrices, can regulate biological responses of cells and in particular adhesion, migration, growth and differentiation which are essential parameters for tissue integration. These responses have been extensively investigated in interphase cells, but little is known whether and how material elasticity affects mitotic cells. We used polyelectrolyte multilayer films as model substrates with elastic modulus ranging from Eap = 0 up to Eap = 500 kPa and mitotic PtK2 epithelial cells to address these important questions. Soft substrates (Eap < 50 kPa) led to abnormal morphology in chromosome segregation, materialized by chromatin bridges and chromosome lagging. Frequency of these damages increased with decreasing substrate stiffness and was correlated with a pro-apoptotic phenotype. Mitotic spindle was not observed on soft substrates where formation of chromatin damages is due to low β1-integrin engagement and decrease of Rac1 activities. This work constitutes the first evidence that soft substrates hinder epithelial cell division. In perspective, our findings emphasize the prime incidence of the material elasticity on the fate of the phenotype, especially of stem cells in the mitotic phase.     

Functional analysis of Cobra Venom Factor/human C3 chimeras transiently expressed in mammalian cells

The complement activating venom component Cobra Venom Factor (CVF), a functional and structural homologue of the human complement component C3, forms a stable CVF-dependent C3 convertase complex, which, in contrast to C3-dependent convertase effects continuous activation of the complement and, thereby, decomplementation. In order to elucidate the mechanism underlying the enhanced activity of CVF compared to human C3, we generated two CVF/C3 chimeras and established different affinity-based assay systems for functional analysis of these constructs. To allow for convenient expression and subsequent functional characterisation, the CVF/C3 chimeras as well as CVF and C3 were transiently expressed in mammalian cells. Problems due to the low concentration of the recombinant proteins in the supernatants of transient expressions were circumvented by fusion to peptide tags enabling their efficient immobilisation onto suitable surfaces and subsequent characterisation. In an alternative approach monoclonal antibody fragments generated from a semisynthetic phage display scFv library were employed for concentrating the recombinant proteins by immunoprecipitation. Utilising both approaches all transiently expressed proteins could be characterised for their complement consumption activity. The data obtained with the CVF/C3 chimeras demonstrate that the increased stability of the CVFBb complex is independent of the domains in CVF corresponding to binding sites of factor B and H and the cleavage sites of factor I in the human C3 molecule.     

N-methyl-D-aspartate and aspartate raise the cytosolic free calcium concentration by acting upon receptors transiently expressed on immature cerebellar Purkinje cells.

N-Methyl-D-aspartate (NMDA) or aspartate (Asp) increased the cytosolic free calcium concentration ([Ca]in) in some populations of Purkinje cells dissociated from immature rat cerebellum. The NMDA- and Asp-induced rise in [Ca]in was affected only a little by adding glycine or NMDA antagonists, but was reduced either by adding Mg2+, Gallopamil hydrochloride (D-600) and gamma-amino-butyric acid, or by removing external Na+. The results suggest that stimulation of the NMDA-sensitive receptors transiently expressed on immature Purkinje cell soma results in a rise in [Ca]in through the activation of voltage-dependent Ca2+ channels.     

Antigen presentation by Toxoplasma-infected cells: antigen entry through cell membrane fusion.

The antigen presentation of cells infected by Toxoplasma gondii (T.g.) for MHC class-I-restricted cytotoxic T cells (CTL) was blocked by treating the T.g.-infected cells with the fungal antibiotic brefeldin A. Electron microscopic analysis of T.g.-infected murine and human-antigen-presenting cells (APC) prepared by the combination of quick-freezing with the deep-etching or freeze-substitution methods revealed that the outer membrane of T.g. fused with some parts of the vacuolar membranes of APC to build up channel-like structures. Work station and cytofluorometry studies detected 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in cells infected by BCECF-labeled T.g., which strongly suggested the direct entry into cells of BCECF derived from T.g. These findings provide evidence that T.g. antigens enter the cytoplasm of APC through cell membrane fusion of T.g. and T.g.-infected APC, and that T.g.-infected APC present T.g. antigen for MHC class-I-restricted CTL through the endogenous pathway of antigen presentation.     

The hemagglutinin-neuraminidase protein of peste des petits ruminants virus is biologically active when transiently expressed in mammalian cells

The genes coding for the surface glycoproteins hemagglutinin-neuraminidase (HN) of the peste des petits ruminants virus (PPRV) and hemagglutinin (H) of rinderpest virus (RPV) were cloned in a cytomagalovirus promoter driven expression vector and expressed transiently in mammalian cells. The protein expression was apparent 24 h after transfection and the expressed proteins were detected at the cell surface. The transiently expressed PPRV HN protein was found to be biologically active in possessing hemadsorption and neuraminidase activities. On the other hand, RPV H protein exhibited neuraminidase activity but was deficient in hemadsorption activity. The substrate specificity of the neuraminidase activity of these two proteins differed distinctly. The presence of neuraminidase activity in both PPRV HN and RPV H proteins is unusual among members of the morbillivirus genus.     

Properties of horizontal cells transiently appearing in the rat dentate gyrus during ontogenesis

In an ontogenetic study, combining morphological analysis and patch clamp recordings, a transiently appearing horizontal cell type was identified in the dentate gyrus. The cells were exclusively located in the outer third of the stratum moleculare. They were present at postnatal day 2 (P2) and could be identified with fluorescent dyes until around P14. The morphology was bipolar, with a putative axonal and a dendritic process stretching out parallel to the pial surface without any preferential direction. Patch clamp studies in the current and voltage clamp mode were performed in hippocampal slices on visually identified horizontal cells, between P4 and P7, which were subsequently stained with lucifer yellow. The cells had a low resting membrane potential, around — 55 mV. They were excitable, displaying broad action potentials (duration 3–20 ms) and, unlike mature dentate granule cells, they also expressed a strong delayed inward rectifier with properties reminiscent of the I_Q current. Unlike granule cells, no postsynaptic signals could be observed during elevation of [K⁺]_o or electrical stimulation, suggesting that the horizontal cells did not participate in functional hippocampal circuitry. We suggest that these cells represent migrating cells with subsequent differentiation to granule cells or inhibitory interneurons. Alternatively they may be part of the early radial glia or serve as transient target cells for afferent fibres between the entorhinal cortex and the dentate gyrus.     

Activation of p38 mitogen-activated protein kinase during normal mitosis in the developing retina

The p38 member of the mitogen-activated protein kinase superfamily is engaged by phosphorylation in response to environmental stress signals, and may have either permissive or inhibitor roles upon cell proliferation. The cell cycle in the proliferative zone of the retina is tightly controlled and proceeds in synchrony with interkinetic migration of the neuroblast nuclei. We examined the association of p38 kinase activity with the cell cycle in the normal, non-stressed retina of the developing rat, maintained either in vivo or in vitro. Using immunohistochemistry, we show that mitotic profiles in the developing retina are highly enriched for phosphorylated p38. Blockade of p38 activity with the chemical inhibitor SB203580 for 4 h transiently arrested cells at the metaphase-anaphase transition and induced cell death after 20 h. p38 inhibition induced an aberrant mitotic profile, with chromosomes arranged in one side of the cell.The data show that p38 is active during normal mitosis and we suggest that p38 is required for the proper cell cycle progression during metaphase-anaphase transition in retinal neuroblasts.