Human Embryo Lung Fibroblast Cells Research Articles

The Role of Oxidative Stress in Hormesis Induced by Sodium Arsenite in Human Embryo Lung Fibroblast (HELF) Cellular Proliferation Model

Hormetic dose-response relationships induced by environmental agents are often characterized by a low-dose stimulation and a high-dose inhibition. The mechanisms underlying hormesis induced by environmental agents still remain an enigma; however, hormetic consequences may have significant implications for health risk assessments. To investigate the role of oxidative stress in hormetic phenomena associated with cell proliferation induced by sodium arsenite, the levels of reactive oxygen species (ROS), lipid peroxidation (LPO), and heat-shock proteins (HSP) and the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were measured in human embryo lung fibroblast (HELF) cells after treatment with sodium arsenite at various concentrations for differing times. Results showed that sodium arsenite induced significant cell proliferation at low concentrations (0.5 μM for 12, 24, and 48 h), but inhibited cell growth at high amounts (5 and 10 μM for 24 and 48 h), reflected as a β concentration-response curve. Data indicated that the relationship between ROS levels and sodium arsenite exposure concentration displayed a positive correlation. It was found out that sodium arsenite at high concentrations induced LPO damage. The activities of SOD were enhanced at low metal concentrations but inhibited with high amounts in a concentration-dependent manner. Similarly, heat-shock protein 27 (HSP27) levels were increased by sodium arsenite of low concentrations with early exposure time (3, 6, and 12 h), but decreased with high metal concentrations with greater exposure time (24 and 48 h).Sodium arsenite decreased HSP70 expression at lower concentrations, but increased HSP70 expression at higher concentration. The results indicated that this cellular hormetic model of cell proliferation induced by sodium arsenite occurred in HELF cells, which may explain contradictory effects seen with this metal. Sodium arsenite at low concentrations induced enhanced ROS generation without cytotoxicity and a cellular protective effect. In contrast, sodium arsenite at high concentrations produced marked ROS formation, marked oxidative stress, and cellular damage, as evidenced by LPO.

Biphasic effect of arsenite on cell proliferation and apoptosis is associated with the activation of JNK and ERK1/2 in human embryo lung fibroblast cells

Biphasic dose-response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high-dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. In the present study, we aimed at investigating the relationship between biphasic effect of arsenite on cell proliferation and apoptosis and activation of JNK and ERK1/2 in human embryo lung fibroblast (HELF) cells. Our results demonstrated that cell proliferation may be stimulated at lower concentrations (0.1 and 0.5 microM) arsenite but inhibited at higher concentrations (5 and 10 microM). When cell apoptosis was used as the endpoint, the concentration-response curves were changed to U-shapes. During stimulation phospho-JNK levels were significantly increased at 3, 6, and 12 h after 0.1 or 0.5 microM arsenite exposure. Phospho-ERK1/2 levels were increased with different concentrations (0.1-10 microM) of arsenite at 6, 12, and 24 h. Blocking of JNK pathway with 20 microM SP600125 or ERK1/2 by 100 microM PD98059 significantly inhibited biphasic effect of arsenite in cells. Data in the present study suggest that activation of JNK and ERK1/2 may be involved in biphasic effect of arsenite when measuring cell proliferation and apoptosis in HELF cells. JNK activation seems to play a more critical role than ERK1/2 activation in the biphasic process.

Inhibition of the pathway of benzo(a)pyrene-induced cell cycle changes by all-trans retinoic acid in lung fibroblast

To investigate the reverse effect of all-trans retinoic acid (ATRA) on Benzo (a) pyrene (B (a) P)-induced cyclin D1, CDK4, E2F-1 and E2F-4 expression and cell cycle progression in human embryo lung fibroblast (HELF). After HELF cells was treated with ATRA, they were exposed to 2 micromol/L of B (a) P. Western blotting was employed to detect protein expression level; the RNA transfection techniques was used to investigate ATRA-induced signal pathway; flow cytometry was used to detect cell cycle progression. After treatment with 2 micromol/L B (a) P for 24 h, the expression of cyclin D1 and E2F-1 were both increased significantly in HELF; the expression of E2F-4 and CDK4 were not changed markedly; pretreatment with 0.1 micromol/L ATRA for 24 h could efficiently decrease B (a) P-induced overexpression of cyclin D1 and E2F-1; stimulation to antisense cyclin D1 or antisense CDK4 by B (a) P could significantly impair E2F-1 up-regulation; pretreatment with ATRA, cells with antisense cyclin D1 or antisense CDK4 showed a less decrease in B (a) P-induced overexpression of E2F-1 compared to similarly treated control cells; flow cytometry analysis showed B (a) P promoted cell cycle progression from G(1) phase to S phase, while pretreatment with ATRA could inhibit B (a) P-induced cell cycle progression by an accumulation of cells in the G(1) phase. ATRA could block B (a) P-induced cell cycle promotion through cyclin D1/E2F-1 pathway in HELF.

Immortalization of human WI38 cells is associated with differential activation of the c-myc origins.

To study the possible relationships between origin activities and cellular processes leading to malignancy, we used an isogenic system of human embryo lung fibroblast cells WI38 and a SV40-transformed variant, WI38 VA13 2RA (WI38(SV40)). We found that the activities of all initiation sites at the c-myc locus were approximately two-fold as high in WI38(SV40) cells as in WI38 cells. Thus, higher initiation frequency of origins at certain loci is induced with cell immortalization, one of the steps in the multi-step process leading to malignancy. We measured the activities of the four c-myc promoters P0, P1, P2, and P3 with nuclear runon assay in the two cell lines in order to detect potential individual promoter changes that may be also associated with immortalization by SV40 virus. The results show that the activities of the promoters P0, P1, and P3 did not significantly change, but the activity of the major promoter P2 in WI38(SV40) cells was about 7.5- to 8.0-fold as high as that in WI38 cells. The increased activity of promoter P2, although approximately 600 bp downstream of one of the major DNA replication initiation sites, had no preferential influence on the major sites of origin activity. Since the distribution of nascent strand abundance was not significantly altered, binding of transcription factors does not seem to facilitate the assembly of pre-replication complex (pre-RC) or otherwise preferentially alter the activities of the DNA replication proteins at this major initiation site.

DNA strand breaks and poly (ADP-ribose) polymerase activation induced by crystalline nickel subsulfide in MRC-5 lung fibroblast cells.

Nickel compounds are potent carcinogens. Their carcinogenicity is believed to be associated with their solubility and cellular uptake. In the present study, we assessed the in vitro genotoxic effect of a water-insoluble nickel compound, crystalline nickel subsulfide (alpha-Ni3S2) on human embryo lung fibroblast cell line (MRC-5 cells). DNA strand breaks was evaluated using single cell gel electrophoresis, or comet assay. The alpha-Ni3S2 induction of poly (ADP-ribose) polymerase (PADPRP), a nuclear enzyme associated with DNA damage and repair was also studied. Hydrogen peroxide (H2O2) was used as a reference compound. A dose-response relationship was found between alpha-Ni2S2 concentrations (2.5 micrograms/cm2 to 20 micrograms/cm2) and the comet tail length. The increase of PADPRP activity of alpha-Ni2S2 treated MRC-5 cells was also significant and dose-dependent within the concentration range of 2.5 micrograms/ cm2 to 10 micrograms/cm2. Close associations have been found between the comet length and PADPRP level for H2O2 (r = 0.98) and alpha-Ni3S2 (r = 0.97). These results clearly suggest that alpha-Ni3S2 is a potent agent in inducing DNA strand breaks, which may be closely related to its carcinogenic effects. Data from the present study also suggest that both comet assay and PADPRP determination are sensitive techniques for quantitative evaluation of DNA damage induced by nickel compounds.

Antiviral activity of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil against thymidine kinase negative strains of varicella-zoster virus.

Mechanism of antiviral activity of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) against the YSR strain of varicella-zoster virus (VZV), which is a mutant derived from the wild YS strain and is completely deficient in viral thymidine kinase (TK), was searched in comparison with antiviral activity of other thymidine analogues, guanosine analogue and thymidylate synthase (TS) inhibitor in human embryo lung fibroblast cells. Thymidine analogues, such as BV-araU,5-iododeoxyuridine (IUDR), 1-beta-D-arabinofuranosylthymine (araT), and guanosine analogue, such as 9-(2-hydroxyethoxymethyl)guanine (ACV), showed higher antiviral activity to the YS strain than to the YSR strain. Though, BV-araU also had the antiviral activity of a microgram level against the YSR strain. In contrast to these results, TS inhibitor, 5-fluorodeoxyuridine (FUDR), had higher antiviral activity to the YSR strain than to the YS strain. Highly synergistic antiviral activities of FUDR to the YS strain and the YSR strain were observed in combination with IUDR, araT, or ACV. However, weakly synergistic or additive inhibition to the YSR strain was shown in combination of BV-araU and FUDR, in spite of highly synergistic effect of this combination to the YS strain. The viral and cellular TS activity was partially inhibited by BV-araU monophosphate, but not by BV-araU. These results indicate that BV-araU is converted into BV-araU monophosphate by cellular TK, and the inhibition of TS activity by BV-araU monophosphate in the YSR strain-infected cells results in the suppression of viral replication.

Effects of various nucleosides on antiviral activity and metabolism of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil against herpes simplex virus types 1 and 2.

Uptake of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) into herpes simplex virus type 1 (HSV-1)- and 2 (HSV-2)-infected cells was elevated about 190 to 40 times, compared with that into mock-infected human embryo lung fibroblast cells. Uptake was not enhanced by infection with thymidine kinase-negative HSV-1 and HSV-2 mutants, however. In HSV-1-infected cells, 9.7% of BV-araU was phosphorylated to BV-araU triphosphate, but only 1.1% was phosphorylated in HSV-2-infected cells. The antiviral effect, uptake, and turnover of BV-araU were inhibited significantly by thymidine (dThd), moderately by deoxyuridine, and not at all by deoxycytidine. On the other hand, the antiviral activity of acyclovir (ACV) was inhibited only by dThd. The effect of BV-araU was influenced by dThd and dThd phosphates (mono-, di-, and triphosphates), and the effect of ACV was influenced only by dThd, which competitively inhibited the phosphorylation of ACV to ACV monophosphate. The combination of 5-fluorodeoxyuridine (FUdR)-BV-araU or FUdR-ACV had a synergistic effect on HSV-1 and HSV-2 replication. The effect of FUdR on the turnover of BV-araU in HSV-1- and HSV-2-infected cells was analyzed by high-performance liquid chromatography. In HSV-1-infected cells, 86% of the BV-araU was phosphorylated to BV-araU triphosphate, but no effect was observed in HSV-2-infected cells, in which 98% of the BV-araU remained as BV-araU monophosphate.

Efficacies of antiherpesvirus nucleosides against two strains of herpes simplex virus type 1 in Vero and human embryo lung fibroblast cells.

Antiviral activities of five nucleoside analogs against the VR-3 and WT-34 strains of herpes simplex virus type 1 (HSV-1) were investigated in Vero and human embryo lung fibroblast (HEL) cells. In HEL cells, the compounds showed antiviral activities against both strains of HSV-1, but in Vero cells, the antiviral activities of the compounds were reduced in proportion to their antiviral indexes (the 50% inhibitory dose [ID50] for cell growth divided by the 50% plaque reduction dose for virus). The ratio of the ID50 in Vero cells to the ID50 in HEL cells was larger in VR-3-infected cells than in WT-34-infected cells. The following results were obtained. (i) Thymidine kinase (TK; EC 2.7.1.21) activity in the VR-3- or WT-34-infected Vero cells was about half that in VR-3- or WT-34-infected HEL cells. Induction of viral TK was especially low in the VR-3-infected Vero cells. (ii) The ID50 of the plaque reduction assay in hypoxanthine, aminopterin, and thymidine medium revealed that the activity of cellular thymidylate synthetase (EC 2.1.1.45) was important in viral replication in VR-3-infected Vero cells. (iii) The VR-3-infected cells required larger thymidine and thymidine phosphate pools for viral replication than the WT-34-infected cells did, although uptake of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil into infected cells was equal for both strains. (iv) In the VR-3-infected Vero cells, the quantity of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil triphosphate was smaller than that in VR-3-infected HEL cells and WT-34-infected Vero and HEL cells.

Analysis of long-term human cytomegalovirus latency in vitro.

Human leukocyte interferon (IFN-alpha) and acyclovir (ACV) have been used to establish human cytomegalovirus (HCMV) latency in infected human embryo lung fibroblast (HEL-F) cells. HCMV latency was maintained for a short interval (less than 9 days) after removal of inhibitors by increasing the incubation temperature. We now report a model system in which HCMV latency has been dramatically extended. HEL-F cells pretreated with IFN-alpha (200 IU/ml) and ACV (300 microM) were infected with a low MOI of HCMV, and treated for 23 days with the same inhibitor combination at 37 degrees. Infectious HCMV and virus antigens were undetectable at the time of inhibitor removal and remained undetectable during continued incubation at 40.5 degrees. A minimum of 0.4% of the cell population, however, contained a virus genome that could be reactivated at the time of inhibitor removal; this value declined to 0.0005% after 77 days at 40.5 degrees. HCMV reactivation was achieved by maintaining the infected cells at 37 degrees after inhibitor removal or by decreasing the incubation temperature to 37 degrees at any time during maintenance at 40.5 degrees. The HCMV genome was analyzed by blot hybridization in latently infected cells 23 days after inhibitor treatment at 37 degrees or 4 days after inhibitor removal at 40.5 degrees. Although many HCMV-unique DNA genomic sequences were retained in HEL-F cultures after 23 days of inhibitor treatment, a significant reduction in retained HCMV sequences occurred after inhibitor removal and temperature shift to 40.5 degrees. The XbaI HCMV DNA fragments retained in the latently infected HEL-F cultures were present at a copy number of at least 0.5 copies per haploid cell genome equivalent.

Analysis of the herpes simplex virus genome during in vitro latency in human diploid fibroblasts and rat sensory neurons.

We have previously designed in vitro model systems to characterize the herpes simplex virus type 1 (HSV-1) genome during in vitro virus latency. Latency was established by treatment of infected human embryo lung fibroblast (HEL-F) cells or rat fetal neurons with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and was maintained by increasing the incubation temperature after inhibitor removal. Virus was reactivated by reducing the incubation temperature. We have now examined the HSV-1-specific DNA content of latently infected HEL-F cells and rat fetal neurons treated with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and increased temperature. The HEL-F cell population contained, on an average, between 0.25 and 0.5 copies of most, if not all, HSV-1 HindIII and XbaI DNA fragments per haploid cell genome equivalent. In contrast, the latently infected neurons contained, on an average, 8 to 10 copies per haploid cell genome equivalent of most HSV-1 BamHI DNA fragments. There was no detectable alteration in size or molarity of the HSV-1 terminal or junction DNA fragments obtained by HindIII, XbaI, or BamHI digestion of the latently infected neuron or HEL-F cell DNA, as compared with digestion of a reconstruction mixture of purified HSV-1 virion and HEL-F cell DNAs. These data suggest that the predominant form of the HSV-1 genome in either latently infected cell population is nonintegrated, linear, and nonconcatameric.

Use of human embryo lung fibroblasts to detect a heat labile toxin of Escherichia coli from children.

In order to detect the heat labile toxin of Escherichia coli human embryo lung fibroblast cells were seeded with whole cell lysate preparations of the organism to be tested. Positive results consisted of growth inhibition and cytopathic change which were easily seen. Heat-labile toxin was produced by strains belonging to the conventional epidemic serotypes of E. coli (EEC) and by non-EEC strains. Toxin-producing organisms were detected in 20% of healthy children examined. The method is suitable for use in the diagnostic laboratory and is easily adapted to a microtitre plate system suitable for screening purposes.