Adenovirus-mediated wild-type-p53-gene expression sensitizes TNF-resistant tumor cells to TNF-induced cytotoxicity by altering the cellular redox state.

We have shown that the loss of p53 function contributed to resistance of tumor cells to TNF-induced cytotoxicity. In the present study, we evaluated the effect of wild-type p53 (wt-p53) expression on TNF sensitivity, by introducing wt-p53 into MCF7/Adr cells in which p53 was deleted, via a recombinant adenovirus encoding p53 (Ad-p53). Our results indicate that infection with Ad-p53 (50-100 viral particles per cell) resulted in pronounced cytotoxicity, whereas infection with 10 viral particles per cell, which was weakly toxic for the MCF7/Adr cells, sensitized these cells to TNF-induced cell death. Moreover, expression of wt-p53 in MCF7/Adr cells induced the production of reactive oxygen intermediates (ROIs) and caused glutathione (GSH) depletion, indicating disturbances in the cellular redox state. Additional treatment of cells with the anti-oxidant and glutathione (GSH) precursor N-acetylcysteine (NAC) resulted in inhibition of p53-induced ROIs production and in partial restoration of intracellular GSH levels, which was associated with the ability of NAC to inhibit p53-modulated TNF-induced cytotoxicity. Interestingly, Ad-p53 was able to inhibit TNF-induced MnSOD mRNA expression in MCF7/Adr cells, which might contribute to the sensitization of cells to the cytotoxic action of TNF. Taken together, our data strongly suggest that wt-p53 expression sensitizes TNF-resistant MCF7 cells with p53 deletion to TNF-induced cell death by a pathway that is dependent on ROIs production.

Methyltransferase inhibitor S-adenosyl-L-homocysteine sensitizes human breast carcinoma MCF7 cells and related TNF-resistant derivatives to TNF-mediated cytotoxicity via the ceramide-independent pathway.

In this study we investigated the signalling requirements for TNF-induced cytotoxicity modulated by the methyltransferase inhibitor S-adenosyl-L-homocysteine (AdoHcy) using the TNF-sensitive human breast carcinoma MCF7 cells and its established TNF-resistant clones (R-A1 and clone 1001). Our data indicate that inhibition of methylation reactions by adenosine plus homocysteine, which are known to condense within cells to AdoHcy, markedly potentiated TNF-induced cytotoxicity in MCF7 cells and rendered related TNF-resistant variants, TNF-sensitive by a mechanism independent from the ceramide pathway. We demonstrated that the dominant-negative derivative of FADD (FADD-DN) blocked methylation inhibition/TNF-induced cell death. Moreover, TNF-mediated cytotoxicity modulated by AdoHcy was blocked by the ICE-inhibiting peptide z-VAD-fmk, suggesting that an ICE-like protease is required for the methylation inhibition/TNF-inducible death pathway. In conclusion, these results suggest that the methyltransferase inhibitor AdoHcy potentiates TNF-induced cytotoxicity in MCF7 cells and renders TNF-resistant MCF7 clones, TNF-sensitive via the ceramide independent pathway and that FADD and the ICE-like protease are likely necessary components in transducing methylation inhibition/TNF signals for cell death.

Sphingosine-1-phosphate mobilizes intracellular calcium and activates transcription factor NF-kappa B in U937 cells.

Sphingosine-1-phosphate (SPP), a metabolite of sphingolipids, has been implicated as a second messenger in cell growth regulation and signal transduction via calcium mobilization from internal stores. This study shows that SPP mobilizes intracellular calcium in U937 cells and demonstrates for the first time the ability of SPP to activate the transcription factor NF-kappa B in these cells. Furthermore, calcium release from the internal stores by thapsigargin (TG), an inhibitor of the endoplasmic reticulum Ca2+ pump, was associated with activation of NF-kappa B. Moreover, we have shown that while an intracellular calcium chelator BAPTA/AM was able to inhibit both SPP- and TG-induced NF-kappa B activation, it had no effect on TNF-induced NF-kappa B activation. In addition, SPP-induced NF-kappa B activation was blocked both by cyclosporin A, known to inhibit calcineurin phosphatase activity, and by the antioxidant butylated hydroxyanisole. These observations suggest that intracellular calcium mobilization is required for SPP-induced NF-kappa B activation, which may involve calcineurin- and redox-dependent mechanisms.

Iron chelation decreases human immunodeficiency virus-1 Tat potentiated tumor necrosis factor-induced NF-kappa B activation in Jurkat cells.

TNF-alpha stimulates HIV-1 replication via activation of the transcription factor NF-kappa B. TNF-mediated activation of NF-kappa B is known to involve the intracellular formation of reactive oxygen intermediates (ROIs). We recently demonstrated that HIV-1 Tat protein potentiates TNF-induced NF-kappa B activation by downregulation of manganese-dependent superoxide dismutase (MnSOD), shifting the cellular redox state towards pro-oxidative conditions. This study shows that treatment of Jurkat cells with iron chelator deferoxamine (DFO) strongly decreases HIV-1 Tat-potentiated TNF-induced NF-kappa B activation but does not modify NF-kappa B activation by TNF-alpha. The ability of iron chelators to reduce Tat-potentiated TNF-induced NF-kappa B binding activity suggests that iron and intracellular hydroxyl radicals (OH.) are required for Tat effect. Moreover, we have shown that exogenously generated OH. markedly enhanced TNF-induced NF-kappa B activation in a dose-dependent manner while was not sufficient to trigger activation of NF-kappa B by itself. In addition, iron chelators had no effect either on MnSOD activity or on the decrease of this activity by Tat. Iron chelators had also no effect on the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), but could elevate the GSH:GSSG ratio decreased by Tat protein. These observations suggest that the formation of intracellular OH. in the presence of iron ions play a major role in HIV-1 Tat enhancement of TNF-induced NF-kappa B activation and that iron chelation may protect Jurkat T cells, at least in part, against oxidative stress induced by Tat.

HIV type 1 glycoprotein 120 amplifies tumor necrosis factor-induced NF-kappa B activation in Jurkat cells.

This article demonstrates that human immunodeficiency virus type 1 (HIV-1) gp120 amplifies the activity of tumor necrosis factor alpha (TNF-alpha), a cytokine that stimulates HIV-1 replication through activation of NF-kappa B. In CD4-positive Jurkat cells, gp120 potentiates TNF-induced NF-kappa B activation. TNF-mediated activation of NF-kappa B is known to involve the intracellular formation of reactive oxygen intermediates (ROIs). Accordingly, we examined the influence of gp120 on the cellular redox state. We found that gp 120-modulated TNF-induced NK-kappa B activation was inhibited by the antioxidant butylated hydroxyanisole, indicating the involvement of redox-dependent mechanisms. In addition, we showed that gp120 induces intracellular formation of hydrogen peroxide, which is accompanied by a decrease in the ratio of glutathione to glutathione disulfide. In contrast, in the p56lck-deficient J.CaM1.6 T cell line, a derivative of the Jurkat cell line, gp120 was unable to stimulate hydrogen peroxide, to decrease the ratio of GSH to GSSG, and has no effect on TNF-induced NF-kappa B activation. This demonstrated that p56lck protein tyrosine kinase plays an active role in transmitting a signal that increases the oxidative state of the cell and as a consequence amplifies TNF-mediated NF-kappa B DNA binding. We have demonstrated that Tat protein decreased both the Mn-dependent superoxide dismutase (MnSOD) and the cellular glutathione content (GSH). Here we show that, in contrast to Tat, gp120 is unable to inhibit activity and expression of MnSOD and to decrease GSH content. Taken together, our data suggest that gp120 potentiates TNF-induced NF-kappa B activation by stimulating a signal pathway that involves p56lck and the increased formation of reactive oxygen intermediates such as H2O2. These findings may be relevant for the regulation of HIV-1 replication in T cells.

Induction of NO synthesis in macrophages by Newcastle disease virus is associated with activation of nuclear factor-kappa B.

Newcastle disease virus (NDV) has received much attention recently because of its non-specific immune stimulating potential and its various anti-tumor activities. Here we describe that NDV induces synthesis of NO and causes an activation of nuclear factor-kappa B (NF-kappa B) in murine macrophages. These reactions were part of an activation process which included also stimulation of adenosine deaminase and inhibition of 5'-nucleotidase. NDV-mediated NO synthesis and NF-kappa B activation were blocked by an antioxidant (butylated hydroxyanisole), by an inhibitor of protein tyrosine kinase (genistein) and of protein kinase A (H-89), but not by an inhibitor of protein kinase C (staurosporin). These data suggest that signalling requirements of NF-kappa B activation and NO production in NDV-treated macrophages are similar.

HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state.

This study demonstrates that human immunodeficiency virus type 1 (HIV-1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HIV-1 replication through activation of NF-kappa B. In HeLa cells stably transfected with the HIV-1 tat gene (HeLa-tat cells), expression of the Tat protein enhanced both TNF-induced activation of NF-kappa B and TNF-mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein. TNF-mediated activation of NF-kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat-mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV-1 Tat suppressed the expression of Mn-dependent superoxide dismutase (Mn-SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn-SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn-SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat1-72), known to transactivate the HIV-1 long terminal repeat (LTR), no longer affected Mn-SOD expression, the cellular redox state or TNF-mediated cytotoxicity. Thus, our experiments demonstrate that the C-terminal region of HIV-1 Tat is required to suppress Mn-SOD expression and to induce pro-oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

[The functional activity of the urethral neutrophilic granulocytes in patients with chronic urethroprostatitis and its correction]. / Funktsional'naia aktivnost' uretral'nykh neitrofil'nykh granulotsitov u bol'nykh khronicheskim uretroprostatitom i ee korrektsiia.

The functional state of urethral neutrophil granulocytes in patients with chronic urethro-prostatitis, and possibility of miramistin use in complex treatment of the disease were investigated. The data revealed functional suppression of these granulocytes in the patients. Complex treatment with miramistin normalized indices of urethral phagocytic activity, on the contrary, after treatment of the patients without the agent there was no tendency to normalize urethral phagocytic function. The findings suggest that miramistin supplemented to routine antiinflammatory therapy improves curative results in patients with chronic urethro-prostatitis but the drug should be prescribed in accordance with the initial function of urethral neutrophil granulocytes.

[The effect of miramistin on the phagocytic activity of the urethral neutrophilic granulocytes in patients with chronic urethroprostatitis]. / Vliianie miramistina na fagotsitarnuiu aktivnost' uretral'nykh neitrofil'nykh granulotsitov u bol'nykh khronicheskim uretroprostatitom.

It was found that addition of mirastimin to the traditional anti-inflammatory treatment results in stimulation of the absorptive capacity of the urethral phagocytes and improves the results of treatment of patients with chronic urethroprostatitis. The immunomodulator mirastimin should be given with consideration of the initial functional state of urethral neutrophilic granulocytes.

[An immunological analysis of the hemagglutinin from the influenza A virus (H1N1) isolated by using various detergents]. / Immunologicheskii analaiz gemaggliutinina virusa grippa A (H1N1), vydelennogo s pomoshch'iu razlichnykh detergentov.

Immunogenic properties of influenza virus hemagglutinin, isolated by new detergents O-14 (desintegron-O) and B-14 (desintegron-B) have been studied. Hemagglutinin isolated by desintegron-O has been found to be more immunogenic than virions. It has been shown that hemagglutinin isolated by desintegron-B induces a lower humoral immune response than the influenza virus.

[Effect of antimicrobial surface-active agents on the immune response to thymus-dependent antigen in mice]. / Vliianie antimikrobnykh poverkhnostno-aktivnykh veshchestv na immunnyi otvet k timuszavisimomu antigenu u myshei.

The study showed that miramystin, a cationi surfactant, was an immunostimulant inducing an increase in humoral and cellular immunity in response to sheep red blood cells. The observed dose-dependent stimulating effect of miramystin on antibody production and development of DTH recommends its use as an immunologic adjuvant in the laboratory practice. It also indicated to the prospects in investigation of immunologic adjuvant properties of other preparations belonging to surface-active substances.

[Effects of embryonal bone tissue on hemopoiesis]. / Vliianie émbrional'noi kostnoi tkani na hemopoéz.

The influence of human fetal bone (FB) on the hemopoietic and stromal cells were studied in the morphometric assay of the bone marrow trephine biopsy samples after a preliminary cultivation using the Marbrook system for 1-5 weeks. The rat FB effect on the hemopoiesis and survival were also studied in experiment with FB transplantation to rats with prior administration of lethal and sublethal doses of cyclophosphamide. In long-term cultivation of the bone marrow trephine biopsy samples, enhanced proliferation of the fibrous tissue and elimination of hemopoietic elements were seen. Combined cultivation of the trephine biopsy samples of the bone marrow and FB inhibited the fibrous tissue proliferation and enhanced the viability of the hemopoietic and lipid cells. Transplantation of the FB in experiment shortened the leukocytopenia period in rats after sublethal (300 mg/kg) and lethal (500 mg/kg) doses of cyclophosphamide and increased their survival.

[The effect of miramistin on the phagocytic activity of urethral neutrophil granulocytes]. / Vliianie miramistina na fagotsitarnuiu aktivnost' uretral'nykh neitrofil'nykh granulotsitov.

Secondary immunodeficiency, one of whose manifestations is depressed phagocytic activity of urethral neutrophilic granulocytes, develops in patients with chronic urethroprostatitis. Miramistin exerts a dose-dependent stimulating effect on the urethral neutrophilic granulocytes; the maximum stimulating effect is observed when phagocytes are treated with 0.001% solution of the drug.

[Immunostimulating effect of synthetic surface-active agents in experimental tuberculosis]. / Immunostimuliruiushchee deistvie sinteticheskikh poverkhnostno- aktivnykh veshchestv pri eksperimental'nom tuberkuleze.

The influence of synthetic surfactants of different classes (BX-14, O-14, B-14) on an immunologic reactivity of mice with evolving experimental tuberculosis was studied. The use of the substances leads to an increase of the E-rosette forming cells (E-RFC) in the mice thymus gland and spleen, and stimulates the inhibited function of spleen natural killers, without modifying (except the agent O-14) the cytotoxicity level of thymus gland natural killers. The above substances can enhance the absorbing capacity of the spleen macrophages as well as their ability to reduce nitro blue tetrazolium. Attention is drawn to the possibility of using immunostimulators belonging to a group of surfactants to correct immunologic reactivity impairments caused by tuberculosis.

[The rheologic aspects of washed donor erythrocytes]. / Reologicheskie aspekty otmytykh donorskikh éritrotsitov.

Washing off donors' red blood cell pack with NaCl isotonic solution results in improvement of red blood cell rheologic properties: their aggregation reduces, deformability increases, osmotic resistance rises. Heparin addition to the washing off solution in a dose of 5 IU/ml prevents development of antiheparin activity in red blood cells after washing off. Application of washed-off red blood cells of donors has advantages as compared to transfusion of the whole blood.

[Effect of leukocytic interferon on the functional state of bronchoalveolar phagocytes in lung diseases]. / Vliianie leikotsitarnogo interferona na funktsional'noe sostoianie bronkhoal'veoliarnykh fagotsitov pri zabolevaniiakh legkikh.

A study was made of the effect of leukocytic interferon on the phagocytic activity of alveolar macrophages and neutrophils in patients with chronic bronchitis, tuberculosis and sarcoidosis of the lungs. Cells isolated from the patients' bronchoalveolar washing off were used. It was concluded that short-term incubation of macrophages and neutrophils with interferon resulted in phagocytosis enhancement and increased intensity of metabolic processes in them. Interferon produced the most noticeable effect on cells with an initially reduced absorption capacity.