Dominant inhibitory Ras delays Sindbis virus-induced apoptosis in neuronal cells.

Mature neurons are more resistant than dividing cells or differentiating neurons to Sindbis virus-induced apoptotic death. Therefore, we hypothesized that mitogenic signal transduction pathways may influence susceptibility to Sindbis virus-induced apoptosis. Since Ras, a 21-kDa GTP-binding protein, plays an important role in cellular proliferation and neuronal differentiation, we investigated the effect of an inducible dominant inhibitory Ras on Sindbis virus-induced death of a rat pheochromocytoma cell line, PC12 cells. Dexamethasone induction of dominant inhibitory Ras (Ha Ras(Asn17)) expression in transfected PC12 cell lines (MMTV-M17-21 and GSrasDN6 cells) resulted in a marked delay in Sindbis virus-induced apoptosis, compared with infected, uninduced cells. The delay in death after Sindbis virus infection in induced versus uninduced PC12 cells was not associated with differences in viral titers or viral infectivity. No delay in Sindbis virus-induced apoptosis was observed in Ha Ras(Asn17)-transfected PC12 cells if dexamethasone induction was initiated less than 12 h before Sindbis virus infection or in wild-type PC12 cells infected with a chimeric Sindbis virus construct that expresses Ha Ras(Asn17). The delay in Sindbis virus-induced apoptosis in induced Ha Ras(Asn17)-transfected PC12 cells was associated with a decrease in cellular DNA synthesis as measured by 5'-bromo-2'-deoxyuridine incorporation. Thus, in PC12 cells, inducible dominant inhibitory Ras inhibits cellular proliferation and delays Sindbis virus-induced apoptosis. These findings suggest that a Ras-dependent signaling pathway is a determinant of neuronal susceptibility to Sindbis virus-induced apoptosis.     

Recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death.

Influenza virus infection induces apoptosis in cultured cells with an augmented expression of Fas (APO-1/CD95). Caspases, a family of cysteine proteases structurally related to interleukin-1-beta-converting enzyme (ICE), play crucial roles in apoptosis induced by various stimuli, including Fas. However, activation of the caspase-cascade seems to be different in various pathways of apoptotic stimuli. We therefore examined the involvement of caspases in influenza virus-induced apoptosis using caspase inhibitors. We found that z-VAD-fmk and z-IETD-fmk effectively inhibited virus-induced apoptosis, whereas Ac-DEVD-CHO and Ac-YVAD-CHO showed partial and little effect on virus-induced cell death, respectively. Consistently, caspase-3-like activity, but not caspase-1-like activity, was increased in the virus-infected cells. The transfection of plasmids encoding viral inhibitors of caspase (v-FLIP or crmA) into HeLa cells inhibited apoptosis by virus infection. The peptide inhibitors of caspases used in this study did not inhibit viral replication. We conclude that influenza virus infection activates some caspases, and that this activation may be downstream of viral replication.     

Effect of enforced expression of human bcl-2 on Japanese encephalitis virus-induced apoptosis in cultured cells.

Infection by Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis in humans and induces severe cytopathic effects in different types of cultured cells. This study attempted to determine whether apoptosis contributes to virus-induced cell death in a culture system by characterizing JEV lytic infection in baby hamster kidney BHK-21 cells, murine neuroblastoma N18 cells, and human neuronal progenitor NT2 cells. According to our results, the replication of JEV, and not the UV-inactivated virions per se, triggered apoptosis in these cell lines, as evidenced by nuclear condensation, DNA fragmentation ladder, and in situ end labeling of DNA strand breaks with terminal transferase (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). Different strains of JEV, regardless of whether they are neurovirulent to mice, could induce apoptosis of the infected cells. In addition, enforced expression of the human protooncogene bcl-2 in BHK-21 cells, which did not influence virus production, appeared to delay the process of JEV-induced apoptosis, despite the fact that most infected cells were inevitably killed after prolonged cultures. However, Bcl-2 proteins expressed in N18 cells failed to block JEV-induced apoptosis, although they did prevent Sindbis virus-induced apoptosis from occurring in the same cells. This finding suggests that these two viruses may utilize similar but not identical mechanisms to kill their infected cells. The results presented here thus demonstrate that apoptosis can be a general mechanism for JEV-induced cell death and that enforced bcl-2 expression may be inadequate in protecting all cell types from JEV-induced apoptosis in cell cultures.     

Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion

Most squamous epithelial cells are strictly anchorage-dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell-cell interaction, and cell spreading. Treatment with EGF increased cell adhesion-regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion-regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle-associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF-induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion.     

Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA polymerase activity and viral DNA replication by phosphonoacetic acid.

Phosphonoacetic acid specifically inhibited human cytomegalovirus DNA synthesis in virus-infected human fibroblasts as detected by virus-specific nucleic acid hybridization. Inhibition was reversible; viral DNA synthesis resumed upon the removal of the drug. The compound partially inhibited DNA synthesis of host cells in the log phase of growth but had little effect on confluent cells. Studies of partially purified enzymes indicated that phosphonoacetic acid specifically inhibited virus-induced DNA polymerase and had only a slight effect on normal host cell enzymes. The drug was shown to interact directly with virus-induced enzyme but not with the template-primers.     

Proliferation and apoptosis of hippocampal granule cells require local oestrogen synthesis

Ovarian oestrogens have been demonstrated to influence neurogenesis in the dentate gyrus. As considerable amounts of oestrogens are synthesized in hippocampal neurones, we focused on the role of hippocampus-derived estradiol on proliferation and apoptosis of granule cells in vitro. We used hippocampal dispersion cultures, which allowed for cultivation of the cells under steroid- and serum-free conditions and monitoring of oestrogen synthesis. To address the influence of hippocampus-derived estradiol on neurogenesis, we inhibited oestrogen synthesis by treatment of hippocampal cell cultures with letrozole, a specific aromatase inhibitor. Alternatively, we used siRNA against steroidogenic acute regulatory protein (StAR). The number of proliferative cells decreased whereas the number of apoptotic cells increased dose-dependently, in response to reduced estradiol release into the medium after treatment with letrozole. This also held true for siRNA against StAR transfected cell cultures. Application of estradiol to the medium had no effect on proliferation and apoptosis whereas the anti-proliferative and pro-apoptotic effects of StAR knock-down and letrozole treatment were restored by treatment of the cultures with estradiol. Our findings suggest that neurogenesis and apoptosis in the hippocampus require a defined range of estradiol concentrations that is physiologically provided by hippocampal cells but not by gonads.     

Oncolytic vesicular stomatitis virus induces apoptosis via signaling through PKR, Fas, and Daxx

Matrix (M) protein mutants of vesicular stomatitis virus (VSV) are promising oncolytic agents for cancer therapy. Previous research has implicated Fas and PKR in apoptosis induced by other viruses. Here, we show that dominant-negative mutants of Fas and PKR inhibit M protein mutant virus-induced apoptosis. Most previous research has focused on the adapter protein FADD as a necessary transducer of Fas-mediated apoptosis. However, the expression of dominant-negative FADD had little effect on the induction of apoptosis by M protein mutant VSV. Instead, virus-induced apoptosis was inhibited by the expression of a dominant-negative mutant of the adapter protein Daxx. These data indicate that Daxx is more important than FADD for apoptosis induced by M protein mutant VSV. These results show that PKR- and Fas-mediated signaling play important roles in cell death during M protein mutant VSV infection and that Daxx has novel functions in the host response to virus infection by mediating virus-induced apoptosis.     

Moraprenylphosphates suppress reproduction of Taylor murine encephalomyelitis virus and accumulation of VP3 viral protein in susceptible cell cultures BHK-21 and P388D1

Influence of moraprenylphosphates (phosphorylated polyprenol of plant origin) upon the accumulation of Taylor murine encephalomyelitis virus VP3 protein in the susceptible cell cultures was studied. It has been shown that moraprenylphosphates inhibited the accumulation of VP3 at early stages of infectious process. Moraprenylphosphates were found to decrease infectivity of the virus as well as virus-induced cellular apoptosis. Mechanisms of immunomodulating and antiviral activity of moraprenylphosphates and prospects of their use as antiviral drugs have been discussed.     

Protein Kinase Activity of in vitro Cultured Plant Cells in Relation to Growth and Starch Metabolism

Taking tetcyclacis, a norbornenodiazentine derivative, as an example, the influence of a growth retardant on the shoot growth of sunflower, soybean, and maize seedlings grown and treated in hydroculture was investigated. In detail, the reduction in the length of various shoot sections {epicotyl, 1st internode, leaf blade) caused by the retardant was studied. At low concentrations of the retardant (\lt10^-6 M) the shortening effects are substantially attributable to an influence on cell elongation, whereas cell division is inhibited as the concentration increases (τ10^-6 M). A comparison of the effects of tetcyclacis in cell suspension cultures of appropriate plant species showed that also in this system concentrations τ 10^-6 M inhibited cell division growth, i. e. there is comparability of plant/ cell culture regarding the retardant effect on cell division. In contrast to the intact plants, however, cell elongation appears to be of only subordinate importance for the growth of cell cultures, as it has been shown using parsley cell suspension cultures.It is discussed to what extent influencing the gibberellin or sterol biosynthesis by means of tetcyclacis provides an explanation for the concentration-dependent effect on the cell division and cell elongation processes.     

Effects of sex steroids and antihormones on growth, adhesiveness and receptors of L-929 cells cultured in serum-containing and serum-free media.

L-929 cells contain distinct steroid hormone receptors for glucocorticosteroids, for androgens and for estrogens. We studied the effects of different hormones at physiological concentrations on androgen and estrogen receptor protein accumulation and on cell multiplication. The cells were cultured in steroid-free serum-containing medium, either in Petri dishes or in suspension cultures, and in serum-free medium in Petri dishes. The presence of androstanolone (30 nM) in suspension cultures decreased the concentration of estradiol receptor-binding sites in the cytoplasmic fraction. This decrease was progressive following 3, 5 or 10 days of suspension culture in the presence of the androgen; simultaneously a parallel increase in cell multiplication and DNA was observed. The estradiol receptor decrease was approx. 50% after 10 days of treatment and was unaltered after a further 5 days. It was verified that the low androstanolone concentration in the medium did not provoke the translocation of the estradiol receptor into the nucleus. Progesterone 50 nM also decreased the cytoplasmic estradiol binding sites but had no influence on cell growth and no cytoplasmic progesterone receptor could be found. Diethylstilbestrol (30 nM) did not decrease the concentration of androgen receptor.Cell multiplication was stimulated after several days of suspension culture in the presence of either diethylstilbestrol, estradiol or androstanolone at a concentration of 10–30 nM. The specific anti-hormones, tamoxifen and cyproterone acetate, inhibited selectively the growth effects of estrogens and androgen, respectively. L-929 cells could be cultured for a long period of time in serum-free medium in Petri dishes. Cell adhesiveness was increased in the presence of 40 nM androstanolone or 40 nM estradiol, as well as cell multiplication. Dexamethasone had a negative effect on cell adhesiveness and cell growth. The experimental data suggest that at low concentrations the different steroids operated each through its own receptor and were active on cell growth even in serum-free medium.     

Rhabdovirus-induced apoptosis in a fish cell line is inhibited by a human endogenous acid cysteine proteinase inhibitor.

To determine the mechanisms of cell death in rhabdovirus-infected cells, we studied the infection of the epithelial papilloma of carp cell line with spring viremia of carp virus. Studies using electron microscopy, confocal microscopy, and agarose gel electrophoresis revealed changes in cell morphology and DNA fragmentation indicative of apoptosis. The virus-induced apoptosis was inhibited in cells treated with a human endogenous acid cysteine proteinase inhibitor.     

bcl-2 alters influenza virus yield, spread, and hemagglutinin glycosylation.

We previously demonstrated that expression of bcl-2 in Madin-Darby canine kidney (MDCK) cells blocks influenza virus-induced apoptosis and DNA fragmentation. We show here that expression of bcl-2 also reduces the level of infectious virus production and the spread of virus in MDCK cell cultures infected at a low multiplicity of infection. This effect is associated with modified glycosylation of the hemagglutinin protein.     

Dual role of the HIV-1 vpr protein in the modulation of the apoptotic response of T cells

We investigated the effect of vpr, physiologically expressed during the course of an acute HIV-1 infection, on the response of infected cells to apoptotic stimuli as well as on the HIV-induced apoptosis. At 48 h after infection, Jurkat cells exhibited a lower susceptibility to undergo apoptosis with respect to uninfected cells. This effect was not observed following infection with either a vpr-mutated virus or a wild-type strain in the presence of antisense oligodeoxynucleotides targeted at vpr mRNA. Single-cell analysis, aimed at simultaneously identifying apoptotic and infected cells, revealed that resistance to apoptosis correlated with productive infection. Notably, vpr-dependent protection from induced apoptosis was also observed in HIV-1-infected PBMC. In contrast, at later stages of infection, a marked increase in the number of cells spontaneously undergoing apoptosis was detected in infected cultures. This virus-induced apoptosis involved vpr expression and predominantly occurred in productively infected cells. These results indicate that HIV-1 vpr can exert opposite roles in the regulation of apoptosis, which may depend on the level of its intracellular expression at different stages of HIV-1 infection. The dual function of vpr represents a novel mechanism in the complex strategy evolved by HIV to influence the turnover of T lymphocytes leading to either viral persistence or virus release and spreading.     

Anticarcinogenic Alkaloids in the Cultured Cells of Cephalotaxus fortunei

Calli were induced from the leaves and stems of Cephalotaxus fortunei Hook. f. on MS medium supplemented with 0. 1 mg/L KT and 3 mg/L NAA, and from which the suspension culture cell line of this plant was established for the first time. Factors such as light, pH value of the medium, concentration of plant hormone, carbon resources and addition of substances to the medium, which affect the growth of suspension cells were investigated. The results showed that suspension cells grew appropriately at pH 5.8 with a low concentration of sucrose or glucose, and a low level of NAA. No difference effect on cell growth was seen between sucrose and glucose. Phenylalanine and protein hydrolysate were not suitable for cell growth in suspension cultures, and light inhibited cell growth. A sensitive and rapid high-performance liquid chromatographic method has been developed for detecting the alkaloids in cultured cells. The results revealed the following contents of cephalotaxine and its anticancer esters in cultured cells: harringtonine, isoharringtonine and homoharringtonine. The total alkaloid production in cell suspension cultures was doubled as that in solid cultures. The relative amounts of cephalotaxine, drupacine, harringtonine, homoharringtonine and isoharringtonine in suspension cells was 22%, 6%, 8%, 23% and 41% respectively. In addition, other alkaloid as deoxyharringtonine and some steroids, including ergdst-5-en-3-ol. stigmasta-5, 22-dien-3-ol, β-sitosterin and 2-naphthalenamine have also been detected in cell cultures using GC/MS combined technique.     

Control of cell type proportions by a secreted factor in Dictyostelium discoideum

It has been shown that, in Dictyostelium discoideum, conversion of prestalk cells to prespore cells in suspension cultures is inhibited by coexisting prespore cells. To examine whether the inhibition of conversion requires direct cell contact or is mediated by substances secreted by the cells, prestalk cells and prespore cells were incubated in shaken suspension, separated from each other by a dialysis membrane, and conversion of the prestalk cells to prespore cells scored after 24 h. Prestalk-to-prespore conversion was significantly inhibited if the density of the prespore cells was sufficiently high. In contrast, prestalk cells had little influence on prestalk-to-prespore conversion. Media conditioned by prespore cells, but not by prestalk cells, also inhibited the conversion of prestalk cells. Adenosine, propionate, diethylstilboestrol and differentiation inducing factor (DIF), all of which are known to influence the prestalk/prespore differentiation, were examined for their effects on prestalk-to-prespore conversion. Among these, all except adenosine significantly inhibited the conversion. Based on these results, possible mechanisms for maintenance of the constant cell-type ratio in D. discoideum slugs were discussed.     

Selective inhibitory effect of Hu-IFN-gamma on the agarose clonability of tumor-derived lymphoid cell lines

Recombinant human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) were compared for their ability to influence the proliferative capacity of tumor-derived cell lines and of normal B lymphocytes infected in vitro by Epstein-Barr virus (EBV). EBV-induced B-cell proliferation was suppressed almost completely when 10(2) U/ml IFN-alpha were added to the culture medium while the same dose of IFN-gamma had significantly lower inhibitory activity. The pure IFNs differed in their ability to influence the growth of three Burkitt lymphoma-derived cell lines, Raji, Daudi, and Namalwa, depending on whether the cells were propagated in suspension or in semisolid cultures. IFN-alpha inhibited cell proliferation under both culture conditions with thresholds of sensitivity characteristics for each cell line. In contrast, IFN-gamma had no effect on the growth in suspension but it abolished the clonogenic potential of tumor cell lines in semisolid agarose. The results suggest that the two IFN types may exert their growth inhibitory activity through different mechanisms of action.     

Effect of supplementing terpenoid biosynthetic precursors on the accumulation of bilobalide and ginkgolides in Ginkgo biloba cell cultures

The effect of precursor feeding on the production of bilobalide and ginkgolides was studied with suspension cell cultures of Ginkgo biloba. The precursors greatly influenced the productivity of bilobalide and ginkgolides. Precursor supplementation increased the accumulation of both bilobalide and ginkgolides, and with positive effect on cell growth. The GA accumulation by cell cultures was influenced by precursors upstream in the metabolism, whereas the BB accumulation was under the influence of downstream precursors of the terpenoid biosynthetic pathway. Furthermore, precursor feeding modified the ratios of the BB, GA and GB in cells and cell cultures of G. biloba. The studies also aid in understanding effect of precursor feeding on the bilobalide and ginkgolides biosynthetic pathway.     

氟对体外培养的小鼠睾丸间质细胞增殖和细胞凋亡的影响

目的 观察不同浓度氟化钠对睾丸间质细胞增殖和细胞凋亡的影响,为氟中毒的机制研究提供依据.方法 取体外培养的睾丸间质细胞,胰酶消化后制成单细胞悬液,常规培养,待细胞融合率达80％,且未出现细胞分化时,将细胞分4组,加入不同浓度的氟化钠染毒(0,5,10,20 mg/L)睾丸间质细胞,分别干预0,24,48,72,96,1...     

Changes in protein content per cell during growth of mouse L cells

The relationship between cell density and protein content per cell was examined in monolayer and suspension cultures of mouse L cells. In monolayer cultures, the protein content per cell reached a maximum at 6 h after plating and retained this level for 18 h. Thereafter, the protein content per cell declined gradually during the exponential growth phase and finally returned to the initial level at the stationary phase. The changes were neither due to the effect of trypsinization nor to the exhaustion of the medium. The protein content per cell in a sparse culture was always greater than that in a dense culture for monolayer culture of L cells. In suspension culture the increase of protein content per cell during the lag phase was similar to that found in monolayer cultures. However, the gradual decline of protein content per cell observed during the exponential phase of monolayer cultures was not detected during that of a suspension culture. The results suggest that the decrease of protein content per cell in monolayer cultures may be related to some function of cell plasma membrane which could be inhibited by a cell-to-cell contact.     

A correlation between the effects of calcium on intercellular adhesion and electrostatic repulsion between cells.

Calcium ions enchance the mutual adhesiveness of HeLa cells harvested from suspension cultures in which growth is density inhibited. No significant effect of calcium is observed on the mutual adhesiveness of HeLa cells from fast growing suspension cultures. Agglutinative titration of the cells using poly-L-lysine, mol. wt 15000, shows that calcium ions reduce the strength of the repulsive forces on density inhibited HeLa cells. The agglutination curve of the nonrepulsive fast growing HeLa cells is not significantly modified by the addition of calcium. The results support the conclusion that the effect of calcium on the mutual adhesiveness of density inhibited cells is due to a weakening of the repulsive forces on these cells.