Juana Wietzerbin

Expression of Ia antigens by cultured astrocytes treated with gamma-interferon.

The expression of major histocompatibility complex class II or Ia antigens by neural cells has been investigated by indirect immunofluorescence on dissociated cultures from mouse cerebellum, cerebral cortex and dorsal root ganglia. Ia antigen expression was not detectable under standard culture conditions. However, treatment of mixed cultures from the cerebellum and of astrocyte cultures from the cerebral cortex with gamma-interferon preparations induced expression of Ia antigens on a fraction of the astrocytes. Under the same conditions, Ia+ cells could be observed in dorsal root ganglion cultures.

IFN-β induces serine phosphorylation of Stat-1 in Ewing's sarcoma cells and mediates apoptosis via induction of IRF-1 and activation of caspase-7

Four human cell lines derived from Ewings sarcoma, EW-7, EW-1, COH and ORS, were investigated to establish the effects of human recombinant interferon-α2a and human recombinant interferon-β on cell proliferation and apoptosis. All four cell lines were much more sensitive to the antiproliferative effects of IFN-β than of IFN-α. Analysis of the early signals triggered by IFN-α and IFN-β demonstrated that the two IFNs were similarly effective in inducing tyrosine phosphorylation of the Jak-1 and Tyk-2 kinases and the transcription factors Stat-1 and Stat-2. Interestingly, an additional rapid phosphorylation of Stat-1 on serine was observed after IFN-β treatment, with concomitant activation of p38 mitogen-activated protein kinase. In these cells, Stat-1 Ser727 phosphorylation in response to IFN-β was found to be impaired by p38 MAPkinase inhibitor (SB203580). IFN-β induced the formation of the Interferon Stimulated Gene Factor 3 complex more efficiently than IFN-α, as well as sustained induction of IRF-1, which may account for its greater induction of 2′5′oligo(A)synthetase and greater inhibition of cell proliferation. IFN-β, but not IFN-α, induced apoptosis in wild-type p53 EW-7 and COH cell lines, but not in the mutated p53 EW-1 or ORS cell lines. The apoptosis induced by IFN-β in EW-7 and COH cell lines appeared to be mediated by IRF-1 and involved the activation of caspase-7. Ectopic expression of IRF-1 induced apoptosis in all four cell lines which correlated with the activation of caspase-7 and with the downregulation of the Bcl-2 oncoprotein, as observed for IFN-β-induced apoptosis in parental EW-7 and COH cell lines.

Abnormal lymphokine production: a novel feature of the genetic disease Fanconi anemia

SummaryThe correction of chromosomal hypersensitivity to mitomycin C (MMC) in Fanconi anemia (FA) human lymphoblasts is observed by growth in a medium conditioned by normal human cells. Under the same conditions, the cytotoxic effect of MMC on FA cells is restored to an almost normal level. The addition of interleukin-6 (IL-6) to an unconditioned culture medium increased the resistance of FA cells to MMC cytotoxicity. This correcting effect is partially abolished by addition of an anti-IL-6 antibody to the conditioned medium. Both lymphoblasts and fibroblasts derived from FA patients demonstrate a reduction in IL-6 production. Moreover, this lymphokine is not induced by tumor necrosis factors α and β (TNFα and TNFβ) in FA cells, as is the case in normal cells. It is suggested that the observed deficiency in IL-6 production may account for one of the major characteristics of FA disease, i.e., the defect in differentiation of the hematopoietic system.

2-Methoxyestradiol induces apoptosis in Ewing sarcoma cells through mitochondrial hydrogen peroxide production

The Ewing sarcoma is the second most common bone tumor in children and young adults. Despite the advances in therapy, the 5-year survival rate for patients with metastatic disease is poor, indicating the need for alternative treatments. Here, we report that 2-methoxy-estradiol (2-Me), a natural estrogen metabolite, induced a caspase-dependent apoptosis of Ewing sarcoma-derived cells independently of their p53 status. 2-Me-induced apoptosis occurred through the mitochondrial death pathway as evidenced by reduction of the mitochondrial transmembrane potential, cytochrome c release and caspase-9 activation. Treatment of cells with 2-Me resulted in generation of intracellular H2O2, which occurred earlier than caspase-9 activation. The H2O2-reducing agent Ebselen and the lipid peroxidation inhibitor vitamin E decreased both 2-Me-induced caspase-9 activation and cell death, thus providing evidence for a role of H2O2 and lipid peroxides in the initiation of this process. Rotenone, an inhibitor of the mitochondrial respiratory chain, abolished both apoptosis and H2O2 production, thereby identifying mitochondria as the source of H2O2. Moreover, we observed that treatment of cells with 2-Me or H2O2 induced activation of the c-Jun N-terminal kinase (JNK). Overexpression of a dominant-negative mutant of JNK1 reduced 2-Me-induced apoptosis indicating that JNK participates in this process. Altogether, our results provide evidence that 2-Me triggers apoptosis of Ewing sarcoma cells through induction of a mitochondria redox-dependent mechanism and suggest that this compound or other agents that selectively increase the level of reactive oxygen species may prove useful to the development of novel strategies for treatment of Ewing tumors.

IFN-β Impairs Superoxide-Dependent Parasite Killing in Human Macrophages: Evidence for a Deleterious Role of SOD1 in Cutaneous Leishmaniasis

Type I IFNs (IFN-α/β) have only recently gained considerable attention as immunomodulators in nonviral infectious diseases. IFN-β has been shown to protect, in a NO-dependent manner, against murine Old World leishmaniasis caused by Leishmania major, but data in New World leishmaniasis are lacking. We found that IFN-β dose-dependently increases parasite burden in Leishmania amazonensis- as well as Leishmania braziliensis-infected human macrophages, independent of endogenous or exogenous NO. However, IFN-β significantly reduced superoxide release in Leishmania-infected as well as uninfected human macrophages. This decrease in superoxide production was paralleled by a significant IFN-β-mediated increase in superoxide dismutase 1 (SOD1) protein levels. Additionally, IFN-β inhibition of leishmanicidal activity was mimicked by SOD1 and antagonized by either pharmacological or small interfering RNA-mediated inhibition of SOD1. Finally, pronounced SOD1 expression in situ was demonstrated in biopsies from New World cutaneous leishmaniasis patients. These findings reveal a hitherto unknown IFN-β/SOD1 axis in Leishmania infection and suggest that inhibition of SOD-associated pathways could serve as strategy in the treatment of L. amazonensis as well as L. braziliensis infection, major human pathogens.

Strong inhibition of Ewing tumor xenograft growth by combination of human interferon-alpha or interferon-beta with ifosfamide

Ewing sarcoma is the second most common bone tumor in childhood. Despite aggressive chemotherapy and radiotherapy strategies, the prognosis of patients with metastatic disease remains poor. We have recently reported that Ewing tumor cell proliferation was strongly inhibited by IFN-β and to a lesser degree by IFN-α. Moreover, under IFN-β treatment, some cell lines undergo apoptosis. Since the possibility of using IFNs for Ewing tumor treatments may be of interest, we have evaluated the efficacy of Hu-IFNs in a nude mice model of Ewing tumor xenografts. The results reported here show that human type I IFNs, Hu-IFN-α and Hu-IFN-β impaired tumor xenograft take and displayed an anti-growth effect toward established xenografts. Furthermore, we have also shown that combined therapy with Hu-IFNs and ifosfamide (IFO), an alkylating agent widely used in high-dose chemotherapy of Ewing tumors, results in a strong antitumor effect. Pathological analysis showed that Hu-IFN-α/IFO and Hu-IFN-β/IFO were characterized by a dramatic decrease in the mitotic index and marked necrosis, as well as extensive fibrosis associated with numerous calcifications. To our knowledge, this is the first demonstration of a potential antitumor effect of human type I IFNs and IFO on Ewing tumors, providing a rational foundation for a promising therapeutic approach to Ewing sarcoma.

NF-κB activation prevents apoptotic oxidative stress via an increase of both thioredoxin and MnSOD levels in TNFα-treated Ewing sarcoma cells

Repression of activation of c‐Jun N‐terminal kinase (JNK) participates in the anti‐apoptotic effect of nuclear factor‐κB (NF‐κB) in TNFα‐treated Ewing sarcoma cells. As oxidative stress is one of the most prominent activators of JNK, we investigated the relationship between TNFα‐induced NF‐κB activation and the control of oxidative stress. Inhibition of NF‐κB activation resulted in an increase in TNFα‐induced ROS production, lipid peroxidation and protein oxidation. Those ROS and lipid peroxides were both involved in TNFα‐induced apoptosis, whereas only ROS elevation triggered sustained JNK activation. TNFα increased the level of two antioxidant enzymes, thioredoxin and manganese superoxide dismutase by an NF‐κB‐dependent mechanism. Inhibition of expression or activity of these enzymes sensitized cells to TNFα‐induced apoptosis, indicating their functional role in protection from cell death. Thus, agents that inhibit activities of these enzymes may prove helpful in the treatment of Ewing tumors.

Production of nitric oxide (NO) in human hydatidosis: Relationship between nitrite production and interferon-γ levels

Human hydatidosis is characterized by a prolonged coexistence of parasite (Echinococcus granulosus) and host without effective rejection. The basis of the immune response of the patient is poorly understood. Previously, we reported the presence of IFN, TNF-alpha and IL-6 activities in the serum of patients with liver and lung hydatidosis. In the present work, we have investigated the production of nitrite (NO2-) in the serum of hydatidic patients carrying hepatic and pulmonary cysts (range 36-300 microM). Our present data show a correlation between the production of nitrite + nitrate (NO2- + NO3-) and that of circulating cytokines IFN and IL-6. In relapsing patients who did not produce IFN and IL-6, the observed serum NO2- concentrations were low (range 10-37.2 microM), as compared to those detected in patients before surgery. Induction of NO synthase in leukocytes from hydatidic patients was induced by stimulating these cells with a specific parasitic antigen, Antigen-5, as assessed by the increased levels of NO3- + NO2- in the range of 60-85 microM for patients with liver hydatidosis, as compared to the 20-25 microM detected in healthy controls. Collectively, our data indicate that NO2- + NO3- levels correlate with IFN levels and immunoreactivity, and overall suggest that IFN-gamma and nitric oxide production together play a role in the host defense mechanisms in human hydatidosis.

Induction of p21Waf1/Cip1 by TNFα requires NF-κB activity and antagonizes apoptosis in Ewing tumor cells

The Ewing family of tumors is characterized by recurrent reciprocal translocations that generate chimeric proteins, either EWS–FLI-1 or EWS–ERG. These proteins are potent transcriptional activators and are responsible for maintaining the oncogenic properties of tumor cells. Since apoptosis appears to be the main mechanism whereby chemotherapy and radiation kill tumor cells, identification of events that can antagonize apoptosis in Ewing tumors is essential for improving their response to conventional therapies. Here, we report that the transcriptional factor NF-κB is a survival factor for Ewing tumor-derived cells. In fact, inhibition of NF-κB activation as a consequence of the overexpression of a degradation-resistant form of IκBα, IκBα (A32/36), sensitized these cells to TNFα-induced killing. Although treatment with TNFα did not modify the cellular expression of Bcl-2, c-IAP1, c-IAP2, p53 and EWS–FLI-1 proteins, it increased p21Waf1/Cip1 levels. This induction required NF-κB activation since it was not observed in the IκBα (A32/36) expressing cells. Moreover, overexpression of p21Waf1/Cip1 in these IκBα (A32/36)-expressing cells, in which NF-κB and consequently p21Waf1/Cip1 are no longer inducible by TNFα, decreased their susceptibility to TNFα-induced killing. Our results therefore identify p21Waf1/Cip1 as a mediator of the antiapoptotic effect of TNFα-induced NF-κB in Ewing tumor cells.

Regulation of CD26/DPPIV gene expression by interferons and retinoic acid in tumor B cells

Interferons (IFNs α, β and γ) and all trans retinoic acid (RA) have the ability to activate genes with GAS sites. We have found that the promoter of CD26/dipeptidylpeptidase IV (DPPIV) contains a consensus GAS site TTCnnnGAA located at bp-35 to -27, and computer analysis confirmed this sequence to be a putative Stat binding site. Consistent with this finding, we show that IFNs and RA rapidly enhanced CD26 gene and protein expression in chronic B lymphocytic leukemia (B-CLL) cells. Immunoblot analyses revealed that unstimulated B-CLL cells expressed detectable levels of serine/tyrosine-phosphorylated Stat1α, and RA and IFN-γ treatment led to increased levels of tyrosine phosphorylation of Stat1α and its nuclear accumulation. As shown by electrophoretic mobility shift assay, RA and IFN-γ increased the binding of a nuclear protein to the GAS-CD26 element. Shift-Western blotting identified Stat1α as the GAS-CD26 binding factor. Augmented levels of CD26 protein in malignant B cells cultured with IFNs or RA coincided with the enhancement of DPPIV activity. Taken together, our results are in favor of the IFN-/RA-mediated upregulation of CD26/DPPIV in B-CLL through the signaling pathway involving Stat1α and the GAS response element of CD26 promoter.