Potentiation of Friend viral leukemogenesis by 9,10-dimethyl-1,2-benzanthracene in two strains of mice

The polycyclic aromatic hydrocarbon, 9,10-dimethyl-1,2-benzanthracene (DMBA) produced malignancy involving the spleen in SJL/J and B10SJF1 mice when injected ip at 500 micrograms per mouse either alone or in combination with threshold doses of Friend leukemia virus (FLV). The mice that received both chemical and virus died significantly sooner than mice that received either chemical or virus alone, and a synergism between DMBA and FLV was demonstrated in both the virus-resistant B10SJF1 hybrids and virus-sensitive SJL/J mice.     

Electron Microscopy of Friend Murine Leukemia Virus in the Mid-Gut of Experimentally Infected Mosquitoes

After 30 and 78 hr, Friend murine leukemia virus (FLV) particles were detected by electron microscopy in the mid-gut lumen of the mosquitoes Aedes aegypti (Linnaeus) and Anopheles stephensi Liston which had fed on leukemia BALB/c mice infected with FLV. Various developmental stages of the virions were observed within and on the surface of ingested blood cells, particularly young erythroblasts, as well as free in the lumen after budding. These preliminary findings indicate that FLV continues to multiply in the mid-gut of these species for at least 3 days despite the action of digestive enzymes. Detailed studies are in progress to determine the fate of FLV in these mosquito species.     

Effects of Friend leukemia virus (FLV) inoculation in F1 mice and differentiation of FLV-induced leukemia

Effects of Friend leukemia virus (FLV) inoculation in F1 specific pathogen free (SPF) mice were examined. Resistance to FLV was dominantly inherited both in F1 hybrid mice (BDF1) (FLV-resistant & FLV-sensitive with polycythemia) and F1 hybrid mice (B6C3F1) (FLV-resistant & FLV-sensitive with anemia). But the population dynamics of the nucleated cell components of F1 mice after FLV inoculation differed from those of FLV-resistant inbred mice. A small number of mature erythroblasts appeared in the peripheral blood of BDF1 mice. In B6C3F1 mice, erythroblastosis with splenomegaly and polycythemia occurred. However, all of these findings in BDF1 and B6C3F1 mice regressed spontaneously. In F1 mice, FLV induced an intermediate reactive pattern of the two patterns that had been induced in the parental strains. The results indicate that FLV may induce leukemia with various degrees of differentiation, according to the genetic difference of the host.     

Enhancement of Friend Leukemia Virus Infection in Mice by Guaroa Virus: Quantitation and Action of Various Oncogenic and Nononcogenic Viruses

Previous studies have indicated that Guaroa virus (GV), an arbovirus, enhanced the replication of Friend leukemia virus (FLV) in mice. To study further the interaction of GV and FLV, different levels of FLV activity were inoculated into mice. At a level of activity capable of producing limited splenic response, coinfection with GV resulted in a marked increase in spleen-foci activity, mean spleen weight, and amount of infectious virus recoverable from plasma and spleen of dually infected mice. Various oncogenic and nononcogenic viruses were also tested for their ability to enhance FLV infection.     

Modulation of ex vivo cytokine production by splenocytes using in vitro combined therapeutics in murine retroviral model.

Altered levels of Type 1 and Type 2 cytokines are important in retrovirus-induced immunosuppression. The combination of immunostimulatory agents with antiviral drugs alters the course of murine retroviral infections. Previously, it was demonstrated that in vitro treatment of noninfected splenocytes and in vivo treatment of Friend leukemia virus (FLV)-infected mice with the combination of azidothymidine (AZT) and methionine enkephalin (MENK) significantly increases Type 1 cytokine levels and decreases Type 2 cytokines compared with treatment with only AZT. In order to study the effect of the time of initiation of immunomodulation on the course of retroviral infections, we examined the kinetics of cytokine production by isolated splenocytes from infected mice. BALB/c mice were infected with FLV, and spleen cells were removed at specified times postinfection (days 1, 3, 7, 10, and 14). Interleukin (IL)-2, interferon (IFN-gamma, IL-4, and IL-10 production by unstimulated or ConA-stimulated splenocytes treated in vitro with AZT, MENK, or AZT + MENK was determined after 48 h. The capacity of the isolated splenocytes to produce the Type 1 cytokines IL-2 and IFN-gamma in response to stimulation with ConA and combination therapy decreased over the course of infection. These results suggest that MENK treatment initiated later in the course of infection is unable to modulate the cytokine profile and would likely be ineffective in altering the course of FLV induced-disease. The results indicate the necessity to initiate antiretroviral therapy early in infection. Such information may be applicable in designing future regimens for HIV-1 infections in humans.     

Fluvastatin Interferes with Hepatitis C Virus Replication via Microtubule Bundling and a Doublecortin-like Kinase-Mediated Mechanism

Hepatitis C virus (HCV)-induced alterations in lipid metabolism and cellular protein expression contribute to viral pathogenesis. The mechanism of pleiotropic actions of cholesterol-lowering drugs, statins, against HCV and multiple cancers are not well understood. We investigated effects of fluvastatin (FLV) on microtubule-associated and cancer stem cell marker (CSC), doublecortin-like kinase 1 (DCLK1) during HCV-induced hepatocarcinogenesis. HCV replication models, cancer cell lines and normal human hepatocytes were used to investigate the antiviral and antitumor effects of statins. FLV treatment resulted in induction of microtubule bundling, cell-cycle arrest and alterations in cellular DCLK1 distribution in HCV-expressing hepatoma cells. These events adversely affected the survival of liver-derived tumor cells without affecting normal human hepatocytes. FLV downregulated HCV replication in cell culture where the ATP pool and cell viability were not compromised. Pravastatin did not exhibit these effects on HCV replication, microtubules and cancer cells. The levels of miR-122 that regulates liver homeostasis and provides HCV genomic stability remained at steady state whereas DCLK1 mRNA levels were considerably reduced during FLV treatment. We further demonstrated that HCV replication was increased with DCLK1 overexpression. In conclusion, unique effects of FLV on microtubules and their binding partner DCLK1 are likely to contribute to its anti-HCV and antitumor activities in addition to its known inhibitory effects on 3-hydroxy-3-methylglutary-CoA reductase (HMGCR).     

Interspecies determinants of Friend leukemia virus antigens involved in cytolysis of virus-pfoducing cells.

The cytolytic reactivity of a complex goat anti-feline leukemia virus (FeLV) antiserum for mouse cells (Eveline) releasing large quantities of Friend leukemia virus (FLV) was analyzed by the sensitive [14C]nicotinamide release microcytotoxicity assay. Whereas this interspecies killing reactivity could be blocked by absorption of the goat anti-FeLV serum with a preparation of disrupted FLV, absorption with purified FLV gp71, the major envelope glycoprotein, had no effect. Subsequent serum absorptions with the individual FLV structural polypeptides revealed that the lysis of the Eveline cells by the goat anti-FeLV serum is mediated by antibodies recognizing the interspecies determinant of p30, the major internal capsid protein. The expression of this internal viral component at the surface of virus-producing cells is discussed further. The results also demonstrated that removal of approximately 70% of the carbohydrate portion of gp71 with a preparation of glycosidases did not affect the integrity of its interspecies determinant; these results are in agreement with an earlier study (Bolgnesi et al., 1975) that examined primarily the group- and type-specific sites.     

Use of silica to identify host mechanisms involved in suppression of established Friend virus leukemia.

Silica, an agent predominantly toxic for macrophages, inoculated i.v. to Friend leukemia virus (FLV)-infected mice, blocks the FLV-leukemosuppressive effects of chlorite-oxidized oxyamylose (COAM)-statolon treatment. FLV-infected, COAM-statolon-treated mice that have received silica and have failed to suppress FLV leukemia produced normal amounts of interferon, but did not make antibodies cytotoxic for FLV leukemic cells. Transfer of untreated spleen cells, splenic T cells, or thymocytes from mice with suppressed FLV erythroleukemia to FLV-infected mice treated with silica and COAM-statolon restores the humoral immune response to FLV antigens and results in leukemosuppression. Thus, T lymphocytes from mice with suppressed erythroleukemia participate in FLV leukemosuppression either directly as effector cells, or indirectly as helper cell in the production of antibodies to FLV antigens.     

Infection of bone marrow cells in vitro with FLV: Effects on stem cell proliferation,differentiation and leukemogenic capacity

Long-term cultures of proliferating hematopoietic stem cells derived from bone marrow permit the study of the interaction between murine leukemia virus (MuLV) infection and the proliferation and differentiation of stem cells. We have used this system to analyze the replication of different biological variants of MuLV in bone marrow cells; the effect of MuLV infection upon pluripotent stem cell (CFU-S) proliferation; and the effect of MuLV on differentiation of CFU-S along different hematopoietic pathways. Two MuLV variants were studied in detail: the Moloney strain of lymphatic leukemia virus (Mol-MuLV) and the erythroleukemic Friend virus complex (FLV) consisting of the lymphoid leukemia helper virus and the defective spleen focus-forming virus (SFFV). Mol-MuLV and its sarcoma virus pseudotype, MSV(Mol-MuLV), replicate efficiently in the bone marrow cultures; however, CFU-S are lost more readily than in uninfected cultures, and the cultures are soon represented by a majority population of mononuclear macrophages. On the other hand, infection with FLV produces a prolonged survival of the spleen colony-forming cells, CFU-S, and CFU-C (the committed granulocytic precursor cells). Production of erythroleukemogenic SFFV is maintained in these cultures for more than 40 weeks. No erythroblastic differentiation was observed in vitro, however, neither erythroblast precursor cells (CFU-E) nor hemoglobin-producing cells could be detected. This suggests that the target cell for FLV is an earlier precursor cell.     

Role of carbohydrate in biological functions of Friend murine leukemia virus gp71.

Purified gp71 of Friend murine leukemia virus (FLV) can interfere with virus infection, absorb neutralizing antibody, and in the presence of group-specific anti-gp71 antibody, hemagglutinate sheep erythrocytes. Interference by FLV gp71 with several murine leukemia viruses (MuLV) was tested in the XC and S + L- assay systems. Treatment of gp71 with trypsin or Pronase eliminated its interfering capacity. However, treatment with neuraminidase or a mixture of glycosidase enzymes, which left the major serological properties of gp71 intact, did not reduce the interference potential of gp71 for FLV or AKR MuLV. The capacity of gp71 to absorb type- or group-specific virus-neutralizing antibodies was similarly affected by the various enzyme treatments. In contrast, indirect hemagglutination by gp71 was abolished not only by proteases but also by treatment with glycosidase enzymes, although neuraminidase had no effect. Preliminary data indicate that infectivity of FLV or xenotropic MuLV was not affected by short treatment with glycosidase enzymes.     

Rapid virus production and removal as measured with fluorescently labeled viruses as tracers

Pelagic marine viruses have been shown to cause significant mortality of heterotrophic bacteria, cyanobacteria, and phytoplankton. It was previously demonstrated, in nearshore California waters, that viruses contributed to up to 50% of bacterial mortality, comparable to protists. However, in less productive waters, rates of virus production and removal and estimates of virus-mediated bacterial mortality have been difficult to determine. We have measured rates of virus production and removal, in nearshore and offshore California waters, by using fluorescently labeled viruses (FLV) as tracers. Our approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonia and amino acids. The results indicated overall virus removal rates in the dark ranging from 1.8 to 6.2% h(-1) and production rates in the dark ranging from 1.9 to 6.1% h(-1), corresponding to turnover times of virus populations of 1 to 2 days, even in oligotrophic offshore waters. Virus removal rates determined by the FLV tracer method were compared to rates of virus degradation, determined at the same locations by radiolabeling methods, and were similar even though the current FLV method is suitable for only dark incubations. Our results support previous findings that virus impacts on bacterial populations may be more important in some environments and less so in others. This new method can be used to determine rates of virus degradation, production, and turnover in eutrophic, mesotrophic, and oligotrophic waters and will provide important inputs for future investigations of microbial food webs.     

Friend leukemia virus infection enhances DNA damage-induced apoptosis of hematopoietic cells,causing lethal anemia in C3H hosts

Exposure of hematopoietic progenitors to gamma irradiation induces p53-dependent apoptosis. However, host responses to DNA damage are not uniform and can be modified by various factors. Here, we report that a split low-dose total-body irradiation (TBI) (1.5 Gy twice) to the host causes prominent apoptosis in bone marrow cells of Friend leukemia virus (FLV)-infected C3H mice but not in those of FLV-infected DBA mice. In C3H mice, the apoptosis occurs rapidly and progressively in erythroid cells, leading to lethal host anemia, although treatment with FLV alone or TBI alone induced minimal apoptosis in bone marrow cells. A marked accumulation of P53 protein was demonstrated in bone marrow cells from FLV-infected C3H mice 12 h after treatment with TBI. Although a similar accumulation of P53 was also observed in bone marrow cells from FLV-infected DBA mice treated with TBI, the amount appeared to be parallel to that of mice treated with TBI alone and was much lower than that of FLV- plus TBI-treated C3H mice. To determine the association of p53 with the prominent enhancement of apoptosis in FLV- plus TBI-treated C3H mice, p53 knockout mice of the C3H background (C3H p53(-/-)) were infected with FLV and treated with TBI. As expected, p53 knockout mice exhibited a very low frequency of apoptosis in the bone marrow after treatment with FLV plus TBI. Further, C3H p53(-/-) --> C3H p53(+/+) bone marrow chimeric mice treated with FLV plus TBI survived even longer than the chimeras treated with FLV alone. These findings indicate that infection with FLV strongly enhances radiation-induced apoptotic cell death of hematopoietic cells in host animals and that the apoptosis occurs through a p53-associated signaling pathway, although the response was not uniform in different host strains.     

Characterization of structural and immunological properties of specific domains of Friend ecotropic and dual-tropic murine leukemia virus gp70s.

A detailed comparison of the gp70 proteins of cloned ecotropic Friend murine leukemia virus (FLV) and dual-tropic Friend mink focus-forming virus (FrMCF) was performed by analyzing the structural and immunological properties of amino- and carboxy-terminal domains of these molecules generated upon controlled trypsinization. The two gp70s gave characteristic fragmentation patterns; the amino-terminal fragments of FrMCF gp70 were smaller than the corresponding fragments of FLV and contained a trypsin site which resulted in a 19,000-dalton amino-terminal fragment not observed for FLV, whereas both molecules yielded an identically sized carboxy-terminal fragment. All amino-terminal fragments of both gp70 molecules contained an endo H-sensitive oligosaccharide chain; for FrMCF, a second endo H-sensitive carbohydrate was present as well at a carboxy-terminal site for approximately 50% of the molecules. Several aspects of the disulfide interactions of the two gp70s were conserved; in both cases the carboxy-terminal fragments were disulfide bonded to p15(E), there were no disulfide bonds between amino- and carboxy-terminal fragments, and the amino-terminal fragments exhibited a significant increase in mobility upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Analysis of the immunoreactivity of the different domains of the proteins by immunoprecipitation of the fragments with antisera prepared against xenotropic murine leukemia virus and feline leukemia virus gp70s indicated major differences in antigenicity for the amino-terminal domains of FLV and FrMCF gp70, whereas the carboxy-terminal domains were immunologically conserved. Similar analyses with antibodies specific for p15(E) and Pr15(E) demonstrate that these components are conserved as well. These data provide direct evidence that p15(E) and the C-terminal gp70 domain of FrMCF gp70 are related to the corresponding regions of the ecotropic FLV parent and indicate that the acquisition of MCF-specific properties is due to the replacement of the ecotropic amino-terminal gp70 domain with sequences related to those of xenotropic gp70s.     

A procedure for the preferential radioiodination of small amounts of protein in the presence of excess lipids.

The chloramine-T, iodine monochloride, and lactoperoxidase radioiodination procedures were evaluated for their ability to label proteins in the presence of large amounts of reactive lipid. Mouse serum very low density lipoproteins (VLDL) (10% protein, 90% lipid) and detergent-disrupted Friend murine leukemia virus (FLV) (60% protein, 33% lipid) were used as model systems. Based on the distribution of label between protein and lipid, as well as the total amount of label incorporated by each procedure, only a modification of the iodine monochloride procedure preferentially labeled proteins to a high specific activity in both VLDL and FLV. The technique, which is described in detail, is technically simple, rapid, and causes no detectable degradation of proteins.     

Minichromosome maintenance 2 bound with retroviral Gp70 is localized to cytoplasm and enhances DNA-damage-induced apoptosis

The interaction of viral proteins with host-cellular proteins elicits the activation of cellular signal transduction pathways and possibly leads to viral pathogenesis as well as cellular biological events. Apoptotic signals induced by DNA-damage are remarkably up-regulated by Friend leukemia virus (FLV) exclusively in C3H hosts; however, the mechanisms underlying the apoptosis enhancement and host-specificity are unknown. Here, we show that C3H mouse-derived hematopoietic cells originally express higher levels of the minichromosome maintenance (MCM) 2 protein than BALB/c- or C57BL/6-deriverd cells, and undergo more frequent apoptosis following doxorubicin-induced DNA-damage in the presence of the FLV envelope protein gp70. Dual transfection with gp70/Mcm2 reproduced doxorubicin-induced apoptosis even in BALB/c-derived 3T3 cells. Immunoprecipitation assays using various deletion mutants of MCM2 revealed that gp70 bound to the nuclear localization signal (NLS) 1 (amino acids 18-24) of MCM2, interfered with the function of NLS2 (amino acids 132-152), and suppressed the normal nuclear-import of MCM2. Cytoplasmic MCM2 reduced the activity of protein phosphatase 2A (PP2A) leading to the subsequent hyperphosphorylation of DNA-dependent protein kinase (DNA-PK). Phosphorylated DNA-PK exhibited elevated kinase activity to phosphorylate P53, thereby up-regulating p53-dependent apoptosis. An apoptosis-enhancing domain was identified in the C-terminal portion (amino acids 703-904) of MCM2. Furthermore, simultaneous treatment with FLV and doxorubicin extended the survival of SCID mice bearing 8047 leukemia cells expressing high levels of MCM2. Thus, depending on its subcellular localization, MCM2 plays different roles. It participates in DNA replication in the nucleus as shown previously, and enhances apoptosis in the cytoplasm.     

Rapid Virus Production and Removal as Measured with Fluorescently Labeled Viruses as Tracers

Pelagic marine viruses have been shown to cause significant mortality of heterotrophic bacteria, cyanobacteria, and phytoplankton. It was previously demonstrated, in nearshore California waters, that viruses contributed to up to 50% of bacterial mortality, comparable to protists. However, in less productive waters, rates of virus production and removal and estimates of virus-mediated bacterial mortality have been difficult to determine. We have measured rates of virus production and removal, in nearshore and offshore California waters, by using fluorescently labeled viruses (FLV) as tracers. Our approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonia and amino acids. The results indicated overall virus removal rates in the dark ranging from 1.8 to 6.2% h⁻¹ and production rates in the dark ranging from 1.9 to 6.1% h⁻¹, corresponding to turnover times of virus populations of 1 to 2 days, even in oligotrophic offshore waters. Virus removal rates determined by the FLV tracer method were compared to rates of virus degradation, determined at the same locations by radiolabeling methods, and were similar even though the current FLV method is suitable for only dark incubations. Our results support previous findings that virus impacts on bacterial populations may be more important in some environments and less so in others. This new method can be used to determine rates of virus degradation, production, and turnover in eutrophic, mesotrophic, and oligotrophic waters and will provide important inputs for future investigations of microbial food webs.     

Pharmacokinetics and distribution of fluvoxamine to the brain in rats under oxidative stress

The effects of oxidative stress (OS) on the pharmacokinetics of fluvoxamine (FLV), particularly on FLV distribution in the plasma, were studied in ferric-nitrilotriacetate-induced OS rat models (OS rats). The study protocol involved a continuous FLV infusion (25.0 μg/kg/min). The resulting mean plasma FLV concentration measured in steady state OS rats was 0.13?±?0.01 μg/mL, which was significantly lower than plasma concentrations measured in control rats (0.19?±?0.01 μg/mL). Moreover, the mean FLV concentration in the OS rat brain (0.51?±?0.08 μg/g) was determined to be approximately half the concentration in control rat brains (0.95?±?0.11 μg/g). The FLV concentrations in both the unbound fraction of plasma and erythrocytes of OS rats were significantly greater than that of control rats. These results suggest the potential attenuation of FLV's pharmacological effects in patients under OS.     

Fluvastatin attenuated the effect of expression of β1 integrin in PAN-treated podocytes by inhibiting reactive oxygen species

It is well accepted that β1 integrin plays a key role in maintaining normal podocytes form and functions; however, its mechanism of the potential protective effect remains unclear. Furthermore, the investigation and understanding of the non-lipid-dependent renal protection of Statins in addition to well-known lipid-lowering effect may provide the therapeutic utility and ultimately improve clinical outcome for patients with renal diseases. In the present study, we investigated the effect and mechanism of fluvastatin (FLV) on the expression of β1 integrin in puromycin aminonucleoside (PAN)-treated podocytes in vitro. Cultured human podocytes were treated with PAN, and/or different concentrations of FLV (1 × 10^?8–1 × 10^?5 mol/l), superoxide dismutase (SOD), or H₂O₂, respectively. The expression of β1 integrin and reactive oxygen species (ROS) in human podocytes under each experimental condition was evaluated by western blot, RT-PCR, and 2′7′-dichlorofluorescein 3′6′-diacetate, respectively. The viability of podocytes was also assessed by MTT colorimetry in the present study. The expression of β1 integrin was significantly decreased, and the synthesis of ROS was significantly increased in podocytes following either PAN or H₂O₂ treatment (p < 0.05). The up-regulation of β1 integrin and down-regulation of ROS were also observed in PAN-treated podocytes following lower concentrations of FLV or SOD treatment (p < 0.05, respectively). The cytotoxicity data derived from MTT assay revealed that lower podocyte viability was found in the presence of higher concentrations of FLV, PAN, or H₂O₂. Lower concentration of FLV or SOD can protect podocytes from being impaired by PAN treatment. FLV attenuated the podocyte injury induced by PAN and increased the production of β1 integrin in human podocytes in vitro. This underlying mechanism of FLV may be through inhibiting the activity of ROS in human podocytes.     

Pharmacokinetics and distribution of fluvoxamine to the brain in rats under oxidative stress

Abstract

The effects of oxidative stress (OS) on the pharmacokinetics of fluvoxamine (FLV), particularly on FLV distribution in the plasma, were studied in ferric-nitrilotriacetate-induced OS rat models (OS rats). The study protocol involved a continuous FLV infusion (25.0 μg/kg/min). The resulting mean plasma FLV concentration measured in steady state OS rats was 0.13?±?0.01 μg/mL, which was significantly lower than plasma concentrations measured in control rats (0.19?±?0.01 μg/mL). Moreover, the mean FLV concentration in the OS rat brain (0.51?±?0.08 μg/g) was determined to be approximately half the concentration in control rat brains (0.95?±?0.11 μg/g). The FLV concentrations in both the unbound fraction of plasma and erythrocytes of OS rats were significantly greater than that of control rats. These results suggest the potential attenuation of FLV's pharmacological effects in patients under OS.