Infectious pancreatic necrosis virus internalization and endocytic organelles in CHSE-214 cells.

The early events that take place during the internalization of infectious pancreatic necrosis virus (IPNV) into Chinook salmon embryo cells (CHSE-214) were analyzed ultrastructurally. Endocytic tracers were employed in order to characterize the organization of endocytic organelles in CHSE-214 cells, as well its relation to the IPNV penetration. Results demonstrate that IPNV appear internalized within vesicular compartments which are located peripherally in CHSE-214 cells. Despite the high rate of infectious multiplicity few virus particles were detected inside the cells. Endocytic tracer labelling of tubulovesicular elements and endosomes of host cells showed a well developed endocytic apparatus. Results suggest that endocytosis may be involved during the initiating events in the productive IPNV infection.     

Enhanced expression of EGFP gene in CHSE-214 cells by an ARS element from mud loach (Misgurnus mizolepis)

The origins of replication are associated with nuclear matrices or are found in close proximity to matrix attachment regions (MARs). In this report, fish MARs were cloned into an autonomously replicating sequence (ARS) cloning vector and were screened for ARS elements in Saccharomyces cerevisiae. Sixteen clones were isolated that were able to grow on the selective plates. In particular, an ARS905 that shows high efficiency among them was selected for this study. Southern hybridization indicated the autonomous replication of the transformation vector containing the ARS905 element. DNA sequences analysis showed that the ARS905 contained two ARS consensus sequences as well as MAR motifs, such as AT tracts, ORI patterns, and ATC tracts. In vitro matrix binding analysis, major matrix binding activity and ARS function coincided in a subfragment of the ARS905. To analyze the effects of ARS905 on expression of a reporter gene, an ARS905(E1158) with ARS activity was inserted into pBaEGFP(+) containing mud loach beta-actin promoter, EGFP as a reporter gene, and SV40 poly(A) signal. The pBaEGFP(+)-ARS905(E1158) was transfected into a fish cell line, CHSE-214. The intensity of EGFP transfected cells was a 7-fold of the control at 11days post-transfection. These results indicate that ARS905 enhances the expression of the EGFP gene and that it should be as a component of expression vectors in further fish biotechnological studies.     

Interferonbeta-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells

We evaluated the changes of metallothionein induction and cellular zinc distribution in HepG2 cells by interferonbeta treatment. Immunohistochemical staining of metallothionein was observed in the cytoplasm and nuclei of hepatocytes; which was observed predominantly in the cells treated with interferon and zinc compared to those with zinc alone, interferon alone or the no-treated control. The cellular zinc level was higher in order of the interferon- and zinc-treated cells, the zinc-alone-treated cells, and the interferon-alone-treated cells. Flow cytometry showed that S-phase population increased in interferon-alone-treated cells and interferon- and zinc-treated cells, but not in zinc-alone-treated ones. Cellular elemental distribution was analyzed using in-air micro-particle induced X-ray emission. In zinc-alone-treated sample, X-ray spectra showed good consistency between the enhanced cellular zinc distribution and the phosphorous map. Localizations of bromine followed by interferon treatment were found accompanying a spatial correlation with the phosphorous map. The samples treated with interferon and zinc showed the marked accumulation of zinc and bromine. Discrete bromine accumulation sites were clearly visible with a strong spatial correlation followed by zinc accumulation. These findings suggest that interferonbeta in combination with zinc predominantly induces metallothionein expression in HepG2 cells. In addition, interferonbeta may promote the translocation of metallothionein-bound zinc from cytoplasm to S-phase nuclei.     

Involvement of iron and iron-catalyzed free radical production in ethanol metabolism and toxicity

Lipoperoxidation, a degradative process of membranous polyunsaturated fatty acids, has been suggested to represent an important mechanism in the pathogenesis of ethanol toxicity on the liver and possibly also on the brain. Catalysis by transition metals, especially iron, is involved in the biosynthesis of free radicals contributing to lipid peroxidation. Although the exact nature of the redox-active iron implicated in this catalysis is still unknown, it has been well established that lipid peroxidation can be prevented in vitro by iron chelators such as desferrioxamine. Deprivation of redox-active iron through desferrioxamine inhibits by about 50% the microsomal oxidation of ethanol in vitro and reduces very significantly in vivo the overall ethanol elimination rate in rats. Administration of desferrioxamine together with ethanol also reduces the ethanol-induced disturbances in the antioxidant defense mechanisms of the hepatocyte. It also reduces in mice both the severity of physical dependence on ethanol and lethality following the acute administration of a narcotic dose of ethanol. Chronic overloading of rats with iron results, on the opposite, in an increased rate of ethanol elimination, although alcohol dehydrogenase and catalase activities are reduced and cytochrome P-450 depleted in the liver of such iron-overloaded animals. The magnitude of the ethanol-induced increase in lipid peroxidation and decrease in the major membranous antioxidant, alpha-tocopherol, is exacerbated in iron-overloaded rats. Several disturbances of iron metabolism have been reported in human alcoholics. Their contribution to ethanol toxicity appears very likely in the case of hepatic siderosis associated with alcohol abuse. Ethanol could however disturb iron metabolism even in the absence of gross abnormalities of the total iron stores. It is suggested that ethanol intoxication could increase cellular redox-active iron, thus contributing to an enhanced steady-state concentration of reactive-free radicals. This oxidative stress would lead to lipoperoxidative damage and cellular injury.     

Genistein increases metallothionein expression in human intestinal cells, Caco-2.

Flavonoids found in common vegetables, fruits, and legumes have been shown to possess antioxidant property. This study is the first to demonstrate that one member of the flavonoid family, genistein, can induce the expression of metallothionein (a metal-binding protein with antioxidant property). We found the effect of genistein to be time- and dose-dependent (10-100 microM). The effect can be observed at both protein and mRNA levels and was synergistic to that of 30 microM zinc. Genistein was shown previously to interact with the estrogen receptor and induce gene expression similar to estrogens at a lower affinity. We thus tested the hypothesis that the effect of genistein on metallothionein expression was mediated through the steroid hormone pathway. We found that various glucocorticoids do not affect metallothionein expression in Caco-2 cells. 17Beta-estradiol at 10-100 microM (concentrations much higher than needed to activate the estrogen response element) induced metallothionein expression in Caco-2 cells. However, a synthetic estrogen, diethylstilbestrol, did not increase metallothionein level at 10 microM. 17Beta-estradiol also did not act synergistically with zinc. Thus, genistein may enhance metallothionein expression through an uncharacterized mechanism. Further studies are needed to delineate the molecular mechanism and to determine whether the expression of other genes is also affected by genistein.     

Involvement of metallothionein and copper in cell proliferation

Metallothionein is a low-molecular weight, cysteine-rich, metal-binding protein which has been implicated in the detoxification of toxic metals (cadmium, mercury), metabolism of zinc and copper, as well as in the scavenging of free radicals. Recent evidence suggests that the protein may also be involved in cell proliferation. Based on the experiments carried out so far, it is assumed that the fundamental role of metallothionein in cell proliferation may be to detoxify and/or transfer copper ions from the cytoplasm to the nucleus at the G₁/S phase, which in turn participate in some way in nuclear DNA synthesis.     

Serum-induced expression of metallothionein isoforms in K-562 cells

The metallothionein (MT) expression was studied in the hematopoietic precursor cell line K-562, after serum deprivation and reconstitution of the cells in medium with 10% (v/v) FCS. Serum deprivation for 72 h markedly downregulated the MT mRNA expression, only the isoforms most abundant in normal K-562 cells were clearly detectable. Within 1-1.5 h after serum supplementation however, a definite induction of MT mRNA was noticed, and all isoforms were induced. Forty-eight hours after serum stimulation, the MT mRNA expression of all isoforms decreased again. Also MT protein levels increased twofold 24 h after serum stimulation. These results suggest that MT has a function in the re-entry of resting cells into the cell cycle, this function however could not be assigned to a specific MT isoform. The induction of MT after serum stimulation was independent of protein synthesis, but dependent on phosphorylation.     

An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C

Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.     

A comparison of bovine serum albumin and chicken ovalbumin as supplements for the serum-free growth of chinook salmon embryo cells,CHSE-214.

Chicken ovalbumin and bovine serum albumin (BSA) were compared as supplements to the basal medium, L-15, for the serum-free cultivation of the Chinook Salmon Embryo cell line, CHSE-214. Unlike L-15 alone, ovalbumin and some commercial BSA preparations allowed cell proliferation and development of confluent monolayer cultures. However, only a fatty acid-free BSA (2%) supported continuous proliferation for two years through approximately 15 subcultivations. For this, subcultivation was achieved with non enzymatic cell dissociation solutions. Also, new serum-free subcultivation techniques were developed that utilized avian egg white trypsin inhibitors to terminate the action of either bovine or cod trypsin. Finally, CHSE-214 were successfully cryopreserved in 2% BSA, allowing all cell cultivation steps to be performed in the absence of FBS.     

Heat shock proteins and Bcl-2 expression and function in relation to the differential hyperthermic sensitivity between leukemic and normal hematopoietic cells

A major problem in autologous stem cell transplantation is the occurrence of relapse by residual neoplastic cells from the graft. The selective toxicity of hyperthermia toward malignant hematopoietic progenitors compared with normal bone marrow cells has been utilized in purging protocols. The underlying mechanism for this selective toxicity has remained unclear. By using normal and leukemic cell line models, we searched for molecular mechanisms underlying this selective toxicity. We found that the differential heat sensitivity could not be explained by differences in the expression or inducibility of Hsp and also not by the overall chaperone capacity of the cells. Despite an apparent similarity in initial heat-induced damage, the leukemic cells underwent heat-induced apoptosis more readily than normal hematopoietic cells. The differences in apoptosis initiation were found at or upstream of cytochrome c release from the mitochondria. Sensitivity to staurosporine-induced apoptosis was similar in all cell lines tested, indicating that the apoptotic pathways were equally functional. The higher sensitivity to heat-induced apoptosis correlated with the level of Bcl-2 protein expression. Moreover, stable overexpression of Bcl-2 protected the most heat sensitive leukemic cells against heat-induced apoptosis. Our data indicate that leukemic cells have a specifically lower threshold for heat damage to initiate and execute apoptosis, which is due to an imbalance in the expression of the Bcl-2 family proteins in favor of the proapoptotic family members.     

High-glucose-induced metallothionein expression in endothelial cells: an endothelin-mediated mechanism

Vascular endothelial cells areconstantly exposed to oxidative stress and must be protected byphysiological responses. In diabetes mellitus, endothelial cellpermeability is impaired and may be increased by high extracellularglucose concentrations. It has been postulated that metallothionein(MT) can protect endothelial cells from oxidative stress with itsincreased expression by cytokines, thrombin, and endothelin (ET)-1. Inthis study, we demonstrate that high glucose concentration can induceMT expression in endothelial cells through a distinct ET-dependentpathway. Exposure of human umbilical vein endothelial cells (HUVEC) toincreasing concentrations of glucose resulted in a rapid dose-dependentincrease in MT-2 and ET-1 mRNA expression. MT expression may be furtheraugmented with addition of ET-1. Preincubation of the cells with thespecific ET_B antagonist BQ-788 blocked MT-2 mRNA expressionmore effectively than the ET_A inhibitor TBC-11251. Highglucose also increased immunoreactive MT protein expression and inducedtranslocation of MT into the perinuclear area. Perinuclear localizationof MT was related to high-glucose-induced reorganization of F-actin filaments. These results demonstrate that an increase in extracellular glucose in HUVEC can lead to a rapid dose-dependent increase in MT-2mRNA expression and to perinuclear localization of MT protein withchanges to the cytoskeleton. These effects are mediated via the ETreceptor-dependent pathway.

     

Lipid raft-dependent endocytosis of metallothionein in HepG2 cells

Human hepatocellular carcinoma (HepG2) cells take up metallothionein (MT) by endocytosis. MT co-localizes with albumin but not with transferrin, indicating uptake via a non-classical pathway rather than via clathrin-mediated endocytosis. A lipid raft-dependent uptake is indicated by pravastatin inhibition of cholesterol synthesis and methyl-beta-cyclodextrin inhibition of cholesterol translocation to the plasma membrane, reducing MT uptake by 29% and 69%, respectively. Subcellular fractionation after MT uptake reveals significant amounts of MT in vesicular fractions including lysosomes but virtually no MT in the cytosol. Metals bound to MT are released into the cytosol, however. The findings define a pathway for cellular metal acquisition. Together with results from other studies demonstrating secretion of MT from different cells and the presence of MT in extracellular fluids, the results suggest a function of MT in intercellular communication.