Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis.

Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 [VP3]; 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. Cleavage of VP3 does not alter viral binding to cell monolayers. In previous electron microscopic studies of infected cell cultures, it has been demonstrated that rotavirus particles enter cells by both endocytosis and direct cell membrane penetration. To determine whether trypsin treatment affected rotavirus internalization, we studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Endocytosis inhibitors (sodium azide, dinitrophenol) and lysosomotropic agents (ammonium chloride, chloroquine) had a limited effect on the entry of infectious virus into cells. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 medicated 51Cr, [14C]choline, and [3H]inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.     

Autocrine stimulation of Nb2 cell proliferation by secreted, but not intracellular, prolactin

We have introduced expression constructs for mouse PRL (mPRL) or a nonsecreted form of mPRL into the PRL-responsive Nb2 rat lymphoma cell line. Cell lines resulting from transfection of Nb2 cells with the wild type mPRL construct synthesize and secrete mPRL. These cells are able to grow independently of added lactogens, and conditioned media and cell extracts from these cultures stimulate the growth of Nb2 cells. In contrast, cells synthesizing the nonsecreted mPRL do not proliferate in the absence of added lactogenic hormones, and conditioned media from these cell cultures do not have PRL-like activity in the Nb2 cell growth assay. PRL protein is detected in these nonsecreting cell lines; however, extracts from these lines are generally unable to stimulate Nb2 cell proliferation. These results indicate that cells can respond in an autocrine fashion to PRL, but that an intracellular form of PRL is unable to activate Nb2 cell growth.     

IFN-gamma, but not Fas, mediates reduction of allergen-induced mucous cell metaplasia by inducing apoptosis

Inflammatory responses induced by allergen exposure cause mucous cell metaplasia (MCM) by differentiation of existing and proliferating epithelial cells into mucus-storing cells. Airway epithelia have various mechanisms that resolve these changes to form normal airway epithelia. In this report, we first investigated the state of mucous cell metaplasia and the mechanisms by which MCM is reduced despite continued exposures to allergen. After 5 days of allergen exposure, extensive MCM had developed but was reduced when allergen challenge was continued for 15 days. During this exposure period, IL-13 levels decreased and IFN-gamma levels increased in the bronchoalveolar lavage fluid. In contrast, IL-13 levels decreased but IFN-gamma was not detected at any time point during the resolution of MCM following cessation of allergen exposure. Instillation of IFN-gamma but not anti-Fas caused accelerated resolution of MCM and MCM was not resolved in Stat1-deficient mice exposed to allergen for 15 days, confirming that IFN-gamma is crucial for reducing MCM during prolonged exposures to allergen. IFN-gamma but not anti-Fas induced apoptotic cell death in proliferating normal human bronchial epithelial cells and in human bronchial epithelial cells from subjects with asthma. The apoptotic effect of IFN-gamma was caspase dependent and was inhibited by IL-13, indicating that the Th2 milieu in asthmatics may maintain MCM by preventing cell death in metaplastic mucous cells. These studies could be useful in the understanding of deficiencies leading to chronicity in airway changes and designing novel therapies to reverse MCM and airway obstruction in asthmatics.     

Exogenous HSP70 becomes cell associated,but not internalized,by stressed arterial smooth muscle cells

Summary Cell death within atherosclerotic plaques leads to necrosis and rupture, resulting in vascular occlusion. We have previously demonstrated that addition of exogenous 70 kDa heat shock protein (HSP70) to arterial smooth muscle cells (aSMCs) in vitro can protect against toxins that may initiate necrosis. To determine whether exogenous HSP70 enters aSMCs or acts from outside cells to preserve viability, cultured rabbit aSMCs were stressed by serum deprivation and treated with fluorescently labeled (7-aminomethyl-4-coumarin-3-acetate) or¹²⁵I-radiolabeled HSP70. Cell-associated HSP70 was analyzed using Western blotting, fluoresence spectroscopy, and gamma counting/autoradiogarphy. Surface binding of HSP70 to aSMCs was differentiated from uptake by using trypsin treatment to degrade non-internalized HSP70. Specificity of HSP70 binding was tested by inhibiting uptake of¹²⁵I-HSP70 with excess unlabeled HSP70 or bovine serum albumin (BSA). The effect of unlabeled exogenous HSP70 on endogenous HSP synthesis was also tested. Exogenous HSP70 increased total cell-associated HSP70 2.9- to 3.6-fold over levels present in unstressed aSMCs. However, <5% of the exogenous HSP70 was trypsin-insensitive, indicating that bound HSP70 was not internalized. Binding of¹²⁵I-HSP70 was inhibited by both unlabeled HSP70 and BSA, implying a non-specific interaction with the plasmalemma. Exogenous HSP70 significantly lowered overall protein synthesis by serum-deprived aSMCs, but it did not specifically inhibit synthesis of endogenous HSPs after heat shock. The results indicate that exogenous HSP70 protects viability of stressed aSMCs through interactions with the cell surface rather than via internalization.     

FcgammaRIIB regulates autoreactive primary antibody-forming cell, but not germinal center B cell, activity

The low-affinity FcR for IgG FcgammaRIIB suppresses the development of IgG autoantibodies and autoimmune disease in normal individuals, but how this effect is mediated is incompletely understood. To investigate this issue, we created FcgammaRIIB-deficient versions of two previously described targeted BCR-transgenic lines of mice that contain follicular B cells with specificity for the hapten arsonate, but with different levels of antinuclear autoantigen reactivity. The primary development and tolerance of both types of B cells were unaltered by the absence of FcgammaRIIB. Moreover, the reduced p-azophenylarsonate-driven germinal center and memory responses characteristic of the highly autoreactive clonotype were not reversed by an intrinsic FcgammaRIIB deficiency. In contrast, the p-azophenylarsonate-driven primary Ab-forming cell responses of both clonotypes were equivalently increased by such a deficiency. In total, our data do not support the idea that FcgammaRIIB directly participates in the action of primary or germinal center tolerance checkpoints. In contrast, this receptor apparently contributes to the prevention of autoimmunity by suppressing the production of autoreactive IgGs from B cells that have breached tolerance checkpoints and entered the Ab-forming cell pathway due to spontaneous, or cross-reactive, Ag-mediated activation.     

Defective antigen presentation by human melanoma cell lines cultured from advanced, but not biologically early, disease

The present studies were undertaken to characterize Ag presentation by cultured human melanoma cell lines. Cell lines established from "biologically early" lesions of malignant melanoma were able to present the soluble Ag tetanus toxoid (TT) to autologous and HLA-DR-matched allogeneic, TT-immune T cell clones. Proliferation of T cell clones in response to Ag presented by primary melanoma peaked on day 2 of culture with Ag. Ag presentation was blocked by pretreatment of TT-pulsed and fixed melanoma cells with mAb against HLA-DR, but not HLA-DQ, HLA-DP, or HLA-ABC. Ag processing and presentation were inhibited by treating the melanoma cells with ammonium chloride. In parallel with previous findings from this laboratory demonstrating the inability of cell lines cultured from "advanced" primary or metastatic melanoma to induce autologous T cell proliferation, such cell lines also failed to present this exogenous Ag despite the presence of cell-surface HLA-class II molecules. Thus, in contrast to the finding in biologically early melanoma, none of the multiple TT-immune, T cell clones from autologous patients or HLA-DR matched donors was able to respond to TT presented by melanoma cells cultured from advanced disease. Co-incubation studies revealed that metastatic melanoma cells did not secrete inhibitory substances during the APC assay, however, they were able to process TT, rendering it "immunogenic" in the presence of fixed, autologous non-T cells. When fixed, autologous melanoma cells were assayed for their ability to present processed Ag; fixed cells of early but not advanced disease were able to present Ag in this setting, indicating that the presenting limb becomes flawed in the evolution of the metastatic phenotype. Finally, studies of chloroquine inhibition of the capacity of melanoma cells derived from early primary disease to stimulate autologous peripheral blood T cells suggest that such cells process and present tumor-associated Ag in the same fashion as the "model" Ag TT.     

Requirement of Erk, but not JNK, for arsenite-induced cell transformation.

Trivalent arsenic (arsenite, As3+) is a human carcinogen, which is associated with cancers of skin, lung, liver, and bladder. However, the mechanism by which arsenite causes cancer is not well understood. In this study, we found that exposure of Cl 41 cells, a well characterized mouse epidermal cell model for tumor promotion, to a low concentration of arsenite (<25 microM) induces cell transformation. Interestingly, arsenite induces Erk phosphorylation and increased Erk activity at doses ranging from 0.8 to 200 microM, while higher doses (more than 50 microM) are required for activation of JNK. Arsenite-induced Erk activation was markedly inhibited by introduction of dominant negative Erk2 into cells, while expression of dominant negative Erk2 did not show inhibition of JNK and MEK1/2. Furthermore, arsenite-induced cell transformation was blocked in cells expressing the dominant negative Erk2. In contrast, overexpression of dominant negative JNK1 was shown to increase cell transformation even though it inhibits arsenite-induced JNK activation. Our results not only show that arsenite induces Erk activation, but also for the first time demonstrates that activation of Erk, but not JNK, by arsenite is required for its effects on cell transformation.     

Antigen presentation by resting B cells. Effectiveness at inducing T cell proliferation is determined by costimulatory signals, not T cell receptor occupancy

Resting B cells stimulated the proliferation of two T cell clones much less efficiently than T cell-depleted low-density APC. In contrast, low-density cells and resting B cells stimulated the clones to produce similar levels of inositol phosphates, a rapid biochemical event dependent only on occupancy of the TCR. The inefficient stimulation of T cell proliferation by resting B cell APC was dramatically improved by the addition of allogeneic low-density accessory cells incapable of being recognized by the TCR on the responding T cells. The results are most consistent with a model where low-density and resting B cell APC display similar amounts of Ag/Ia molecule complexes capable of being recognized by the TCR on the responding T cells but differ in the provision of costimulatory signals that, together with TCR occupancy, are required for IL-2 production.     

Nuclear physics (of the cell,not the atom)

The nucleus is physically distinct from the cytoplasm in ways that suggest new ideas and approaches for interrogating the operation of this organelle. Chemical bond formation and breakage underlie the lives of cells, but as this special issue of Molecular Biology of the Cell attests, the nonchemical aspects of cell nuclei present a new frontier to biologists and biophysicists.     

Regulation of squalene synthetase in human hepatoma cell line Hep G2 by sterols, and not by mevalonate-derived non-sterols

Incubations of Hep G2 cells for 18 h with human low-density lipoprotein (LDL) resulted in a decrease of squalene synthetase activity, whereas heavy high-density lipoprotein (hHDL) stimulated the activity. Simultaneous addition of LDL abolished the hHDL-induced stimulation, indicating that manipulating the regulatory sterol pool within the cells influenced the enzyme activity. Blocking the endogenous cholesterol synthesis either at the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase site with compactin or at the 2,3-oxidosqualene cyclase site with the inhibitor U18666A gave rise to an elevation of the squalene synthetase activity. Simultaneous addition of mevalonate abolished the compactin-induced increase. However, at total blockade of sterol synthesis by 30 microM U18666A, added compactin and/or mevalonate did not change the enzyme activity further. It was concluded that sterols regulate the squalene synthetase activity, whereas, in contrast with the regulation of the HMG-CoA reductase activity in Hep G2 cells, mevalonate-derived non-sterols did not influence this enzyme.     

Tail regression induced by elevated retinoic acid signaling in amphioxus larvae occurs by tissue remodeling,not cell death

The vitamin A derived morphogen retinoic acid (RA) is known to function in the regulation of tissue proliferation and differentiation. Here, we show that exogenous RA applied to late larvae of the invertebrate chordate amphioxus can reverse some differentiated states. Although treatment with the RA antagonist BMS009 has no obvious effect on late larvae of amphioxus, administration of excess RA alters the morphology of the posterior end of the body. The anus closes over, and gut contents accumulate in the hindgut. In addition, the larval tail fin regresses, although little apoptosis takes place. This fin normally consists of columnar epidermal cells, each characterized by a ciliary rootlet running all the way from an apical centriole to the base of the cell and likely contributing substantial cytoskeletal support. After a few days of RA treatment, the rootlet becomes disrupted, and the cell shape changes from columnar to cuboidal. Transmission electron microscopy (TEM) shows fragments of the rootlet in the basal cytoplasm of the cuboidal cell. A major component of the ciliary rootlet in amphioxus is the protein Rootletin, which is encoded by a single AmphiRootletin gene. This gene is highly expressed in the tail epithelial cells of control larvae, but becomes downregulated after about a day of RA treatment, and the breakup of the ciliary rootlet soon follows. The effect of excess RA on these epidermal cells of the larval tail in amphioxus is unlike posterior regression in developing zebrafish, where elevated RA signaling alters connective tissues of mesodermal origin. In contrast, however, the RA‐induced closure of the amphioxus anus has parallels in the RA‐induced caudal regression syndrome of mammals.     

Reduction of radiation-induced cell cycle blocks by caffeine does not necessarily lead to increased cell killing

The effect of caffeine upon the radiosensitivities of three human tumor lines was examined and correlated with its action upon the radiation-induced S-phase and G2-phase blocks. Caffeine was found to reduce at least partially the S-phase and G2-phase blocks in all the cell lines examined but potentiated cytotoxicity in only one of the three tumor lines. That reductions have been demonstrated to occur in the absence of increased cell killing provides supporting evidence for the hypothesis that reductions may not be causal in those cases when potentiation of radiation-induced cytotoxicity is observed in the presence of caffeine.     

Cadherin-mediated cell sorting not determined by binding or adhesion specificity.

Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domains of either human E-cadherin or Xenopus C-cadherin, and the specificity of adhesion was compared with cell-sorting assays. None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence. In addition, all cells exhibited similar strengthening of adhesion on both substrates. However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains. These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.     

Suppression of tumorigenicity,but not invasion,in glioblastoma/HeLa cell hybrids

Somatic cell hybrids between SNB-19 human glioblastoma cells and human D98^OR HeLa parental cells were produced and analyzed for their ability to form tumors in nude mice and to invade reconstituted extracellular matrix (Matrigel). Whereas both the SNB-19 and D98^OR HeLa parental cells form tumors, four of six hybrid lines did not form tumors, even after periods up to six months, suggesting that each cell type can complement the tumorigenicity of the other. SNB-19 cells showed high rates of Matrigel invasion at all cell densities examined, whereas D98^OR HeLa cells showed lower rates of invasion that were further reduced at high cell density. All six hybrid cell lines displayed a combination of these properties: at low cell density, the hybrids showed high rates of invasion, similar to the SNB-19 cells, but the invasion rate diminished at higher cell densities, similar to the D98^OR HeLa cells. Taken together, these results provide new experimental evidence that several distinct genetic changes are involved in generating the tumorigenic and invasive phenotype of glioblastoma cells. © 1995 Wiley-Liss, Inc.     

Adenoviral-induced islet cell cytotoxicity is not counteracted by Bcl-2 overexpression

BACKGROUND: The ability to transfer immunoregulatory, cytoprotective, or anti-apoptotic genes into pancreatic islet cells may allow enhanced resistance against the autoimmune destruction of these cells in type 1 diabetes. We describe here an inducible transduction system for expression of the anti-apoptotic bcl-2 gene in insulin-producing cells as a potential tool for protecting against beta-cell death. MATERIALS AND METHODS: Isolated pancreatic rat islet cells or rat insulinoma (RINm5F) cells were transduced using a progesterone antagonist (RU 486) inducible adenoviral vector system, expressing the bcl-2 gene. Bcl-2 overexpression was measured by Western blot assays and flow cytometry analysis. Following exposure to cytokines or to the mitochondrial uncoupler FCCP, cell survival was determined using fluorescence and electron microscopy, and a colorimetric assay (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]- 2H-tetrazolium-5-carboxanilide [XTT]-based) for cell viability. The mitochondrial membrane potential ((m)) was assessed using the lipophilic cationic membrane potential-sensitive dye JC-1. RESULTS: The adenoviral gene transfer system induced Bcl-2 expression in more than 70% of beta-cells and the protein expression levels were successfully regulated in response to varying concentrations of progesterone antagonist RU 486. Exposure of islet cells to proinflammatory cytokines IL-1beta, TNF-alpha, and IFN-gamma, or to the mitochondrial uncoupler FCCP resulted in disruption of the mitochondrial membrane potential ((m)) and beta-cell death. Bcl-2 overexpression stabilized (m) and prevented cell death in RINm5F cells but not in islet cells. In addition, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis. CONCLUSIONS: The RU 486-regulated adenoviral system can achieve an efficient control of gene transfer at relatively low doses of the adenoviral vector. However, Bcl-2 overexpression in islet cells did not prevent adenoviral- or cytokine-induced toxicity, suggesting that the specific death pathway involved in adenoviral toxicity in beta-cells may bypass the mitochondrial permeability transition event.     

CD8+ T cell activation is governed by TCR-peptide/MHC affinity, not dissociation rate

Binding of peptide/MHC (pMHC) complexes by TCR initiates T cell activation. Despite long interest, the exact relationship between the biochemistry of TCR/pMHC interaction (particularly TCR affinity or ligand off-rate) and T cell responses remains unresolved, because the number of complexes examined in each independent system has been too small to draw a definitive conclusion. To test the current models of T cell activation, we have analyzed the interactions between the mouse P14 TCR and a set of altered peptides based on the lymphocytic choriomeningitis virus epitope gp33-41 sequence bound to mouse class I MHC D(b). pMHC binding, TCR-binding characteristics, CD8+ T cell cytotoxicity, and IFN-gamma production were measured for the peptides. We found affinity correlated well with both cytotoxicity and IFN-gamma production. In contrast, no correlation was observed between any kinetic parameter of TCR-pMHC interaction and cytotoxicity or IFN-gamma production. This study strongly argues for an affinity threshold model of T cell activation.     

Establishment of a tetraploid Meth-A cell line through polyploidization by demecolcine but not by staurosporine, K-252A and paclitaxel

Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.     

Centrosomes, and not nuclei, initiate pole cell formation in Drosophila embryos

An injection of aphidicolin into early Drosophila embryos inhibits DNA synthesis and nuclear division, while centrosome replication and many other aspects of the mitotic cycle continue. If aphidicolin is injected at nuclear cycle 7-8, the normal migration of nuclei to the embryo cortex is completely inhibited. In most of these embryos, however, centrosomes continue to migrate in a coordinated manner to the cortex, where they reorganize tubulin, actin, and the overlying plasma membrane. Remarkably, the centrosomes that migrate to the posterior pole of such embryos initiate pole cell formation in the absence of nuclei. These observations demonstrate that centrosomes alone are able to direct a major reorganization of the cortical cytoskeleton when they arrive at the surface of the embryo. They also suggest that the coordinated movement of nuclei to the embryo cortex is mediated by forces acting on the centrosome rather than on the nucleus itself.