Josiane Sancéau

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.

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.

Differential expression of inducible NO synthase in two murine macrophage cell lines

Although primary macrophages and most murine macrophage cell lines such as RAW 264.7 cells respond to interferon‐γ (IFN‐γ) and/or lipopolysaccharide (LPS) by producing large amounts of nitrite, i.e. the oxidation product of nitric oxide (NO) produced by inducible NO synthase (iNOS), other cell lines like P388.D1 cells do not produce significant amounts. To gain insight into the signalling pathway that leads to the induction of iNOS activity, we compared iNOS expression in RAW 264.7 and P388.D1 cells. We showed that IFN‐γ binds to each cell line with a similar affinity. Furthermore, no differences in iNOS gene structure were detectable by Southern blot analysis. Even though no significant nitrite secretion was found in the supernatant of P388.D1 cells stimulated with IFN‐γ and/or LPS, iNOS mRNA expression was induced. In addition, IFN‐γ induced the interferon regulatory factor‐1 (IRF‐1) gene and activated the binding of this factor to its target sequence in the iNOS gene. This binding was recently shown to be necessary for iNOS expression. However, in P388.D1 cells, we were unable to detect the corresponding iNOS protein. These results indicate a deficiency in P388.D1 cells which appears to be restricted to the signalling pathway controlling iNOS protein synthesis. This deficiency does not affect the overall IFN‐γ biological response, but rather a convergent post‐transcriptional step common to IFN‐γ and LPS.

gamma-Glutamyl transpeptidase expression in Ewing's sarcoma cells: up-regulation by interferons.

The genetic hallmark of Ewings sarcoma family of tumours (ET) is the presence of the translocation t(11;22)(q24;q12), which creates the ET fusion gene, leading to cellular transformation. Five human gamma-glutamyl transpeptidase (gamma-GT) genes are located near the chromosomal translocation in ET. gamma-GT is a major enzyme involved in glutathione homoeostasis. Five human cell lines representative of primary or metastatic tumours were investigated to study whether gamma-GT alterations could occur at the chromosomal breaks and rearrangements in ET. As shown by enzymic assays and FACS analyses, all ET cell lines consistently expressed a functional gamma-GT which however did not discriminate steps of ET progression. As shown previously [Sancéau, Hiscott, Delattre and Wietzerbin (2000) Oncogene 19, 3372-3383], ET cells respond to the antiproliferative effects of interferons (IFNs) type I (alpha and beta) and to a much less degree to IFN type II (gamma). IFN-alpha and -beta arrested cells in the S-phase of the cell cycle. We found an enhancement of gamma-GT mRNA species with IFN-alpha and -beta by reverse transcriptase-PCR analyses. This is reflected by up-regulation of gamma-GT protein, which coincides with the increase in gamma-GT-specific enzymic activity. Similarly, IFNs up-regulate the levels of gamma-GT in another IFN-responsive B cell line. Whether this up-regulation of gamma-GT by IFNs is of physiological relevance to cell behaviour remains to be studied.

Location of enhanced ribonuclease activity and of a phosphoprotein kinase in interferon-treated mengovirus-infected cells

Abstract Infection of interferon-treated L cells by mengovirus increased a ribonuclease activity in a sedimentable fraction known to contain the viral replication complex. The enhanced in vitro phosphorylation of a 67,000 M r protein was also observed in the same fraction.

Expression of extracellular and intracellular human IFN-γ in mouse L cells transformed with the human IFN-γ cDNA gene

Abstract Cotransformation with a plasmid containing a thymidine kinase gene (pTK2) and a plasmid encoding human IFN-γ (pTG11) has been used to establish murine L cell lines expressing human IFN-γ. The HuIFN-γ gene was present in 30% of the tk+ cell lines and some of these secreted low levels of IFN into the culture medium. Two of the clones obtained after transformation were selected for detailed analysis. Clone 1–12 constitutively secreted very low levels of HuIFN-γ in the culture medium. This antiviral activity was characterized by its species specificity and antigenicity as authentic human IFN-γ In contrast, clone 3–47 produced a HuIFN-γ activity which could only be detected intracellularly. This clone was resistant to infection both by Vesicular stomatitis (VSV) and Mengo viruses and contained increased levels of enzymes known to be induced by interferon. Dur results suggest that clone 3–47 produces a non-secreted HuIFN-γ like molecule which is able to trigger an antiviral state in the murine cell independent of the interaction with a specific IFN-γ surface receptor.

RECOVERY BY INTERFERON OF MENGOVIRUS-INDUCED SHUTOFF OF HOST PROTEIN SYNTHESIS*

In preincubated cell-free extracts prepared from interferon-treated cells the translation of exogenous viral or cellular mRNAs is inhibited equally.’,2 It is also known that the addition to such extracts of dsRNA activates at least two specific enzymes, the consequence being the inhibition of translation. (Reviewed by C. Bagli~ni.~] Since dsRNA seems to play a major role in stimulating interferon-dependent enzymes. it was interesting to study a functional system in which the challenge virus would provide dsRNA molecules. We have chosen as model L cells infected with mengovirus. It is known that soon after infection by a picornavirus there is an inhibition of cellular RNA and protein synthesis. Later in infection, the synthesis of DNA is also d e ~ r e a s e d . ~ The inhibition of the synthesis of macromolecules is often referred to as “shutoff.“ We decided to study first, the possible action of interferon (IF] on the establishment of the shutoff. Since the level of shutoff varies with the cell lines and with the multiplicity of infection we established our experimental conditions. (FIGURE 1). Confluent monolayers of L cells were infected with different multiplicities of infection (MOI] of mengovirus; after 4 h r cells were pulse-labeled for 30 min with methionine, lysed with detergents (NP40 and DOC] and the proteins analyzed by slab gel electrophoresis. The first lane to the left shows the pattern of host proteins; in the second lane, with 5 pfu/cell viral proteins are very easily recognized, although cellular proteins are also present. It should be remembered that the viron RNA acts as messenger and it is translated from one single initiation site. The unique translation product is a “polyprotein” of about 270,000 MW. Primary and secondary cleavages of this protein give rise to precursors, and finally capsid and noncapsid stable proteins. With 20 and 50 pfu/cell the shutoff of host protein synthesis is markedly established. The counts of this experiment are shown in FIGURE 2, represented by the filled circles. With 5 pfu/cell, although viral proteins are actively synthesized, the total amount of methionine is already lower than in the control noninfected cells. Both, host and viral proteins contributed to the total counts. With 20 pfu cellular proteins are almost absent, the counts correspond principally to viral proteins. With 50 pfu/cell more viral proteins are synthesized, which explains the higher incorporation of the label. When we repeated the experiment with cells pretreated with 200 units IF/ml. we were unable to detect viral proteins.

Helper activity for antibody synthesis encoded by mRNA extracted from human peripheral blood mononuclear cells

Translation products in Xenopus laevis of mRNA from human peripheral blood mononuclear cells were tested for their capacity to replace T cells in the anti-SRBC response of nude spleen cells. When the starting material came from PHA or FCS-stimulated lymphocytes, the translated lymphokines, displaying such a B cell helper activity were found to be encoded by mRNA sedimenting at 6-7S and 13S on a sucrose density gradient. 6-7S mRNA from control, non-stimulated lymphocytes was also able to code for B cell helper activity. Thorough T-cell depletion of mouse responder cell populations left unchanged the activity of 6-7S mRNA products while preventing that of 13S products. The latter were found to contain IL-2, which suggests that their action on B cells was indirect, mediated by T-cell stimulation.

Protein synthesis in extracts from interferon-treated Mengo virus infected cells.

Abstract We previously showed in intact L cells that interferon treatment did not modify the shut-off of cellular RNA and protein synthesis induced by infection with Mengo virus although viral replication is inhibited (1,2). We have also demonstrated that inhibition of host protein synthesis was not due to degradation of messengers since cellular mRNA could be extracted from interferon-treated infected cells and efficiently translated in a reticulocyte lysate(2). Cellular mRNA was not degraded although 2–5A was present as reported here. We prepared cell-free systems from such cells at a time when cellular shut-off was fully established. The undegraded messengers remained untranslated under cell-free protein synthesis conditions and almost no polysomes were detected. The decreased amount of [ 35 S]Met-tRNA-40S complex observed in these lysates might account for the inhibition of protein synthesis at the level of initiation.