Human Interferon-beta Research Articles

The vesicular stomatitis virus matrix protein inhibits transcription from the human beta interferon promoter.

In cells infected by wild-type (wt) vesicular stomatitis virus (VSV) Indiana, host transcription is severely inhibited. DNA cotransfection studies have implicated the VSV matrix (M) protein in this process (B. L. Black and D. S. Lyles, J. Virol. 66:4058-4064, 1992). The M protein inhibited transcription not only from viral promoters in plasmids but also from the chromosomally integrated human immunodeficiency virus type 1 (HIV-1) provirus promoter (S.-Y. Paik, A. C. Banerjea, G. G. Harmison, C.-J. Chen, and M. Schubert, J. Virol. 69:3529-3537, 1995). In this study, we investigated the effect of wt VSV M protein on expression of a reporter gene under control of a cellular promoter (beta-interferon [IFN-beta] promoter), using double transient transfections in BHK and COS-1 cells. The cellular IFN-beta promoter was as susceptible to the inhibitory effect of the M protein as the viral promoters used previously. Viral proteins N, P, and G had no significant effect on reporter gene expression. The M protein gene from VSV mutant T1026R1, which is defective in host transcription inhibition, was cloned and sequenced, and its effect on reporter gene expression was tested. The mutant M protein had a methionine-to-arginine change at position 51 in the protein sequence and did not inhibit transcription from either the IFN-beta promoter or viral promoters. This VSV mutant is a good inducer of IFN, as opposed to the wt virus, which suppresses IFN induction. These results show that the M protein inhibits transcription from cellular as well as viral promoters and that the M protein does not regulate the IFN promoter any differently from viral promoters. While the M protein may play a role in IFN gene regulation, other viral or cellular factors that provide specificity to the induction process must also be involved.

Tamoxifen enhances interferon-regulated gene expression in breast cancer cells.

The molecular basis for the enhanced growth inhibition of MCF-7 human breast cancer xenografts by a combination of human interferon-beta (IFN-beta) and tamoxifen was investigated. Treatment of MCF-7, MDA-MB-231, and BT-20 cells with the combination of IFN-beta and tamoxifen resulted in enhanced antiproliferative effects in vitro. Treatment with the combination of IFN-beta and tamoxifen enhanced the expression of several IFN-beta-inducible genes in human breast carcinoma cell lines relative to levels induced by IFN-beta alone. Tamoxifen alone did not induce transcription of IFN-stimulated genes (ISGs). Augmentation of ISG expression by the combination of IFN-beta and tamoxifen was noted in breast tumor cell lines irrespective of their functional estrogen receptor (ER) status or their dependence on estradiol for growth, suggesting that upregulation of ISGs was independent of ER status. Enhancement of IFN-stimulated gene expression by tamoxifen occurred at the transcriptional level. Expression of transfected reporter genes under the control of IFN-alpha/beta regulated promoters was also enhanced in IFN-beta and tamoxifen-treated cells. Similarly, transcriptional induction of chimeric reporter plasmids driven by an IFN-gamma inducible promoter (GAS; IFN-gamma activated site) was also enhanced by the combination of IFN-gamma and tamoxifen. In tamoxifen treated cells, IFN-beta and IFN-gamma readily activated transcription factors ISGF-3 and GAF, respectively. Therefore, augmentation of ISG expression by tamoxifen is an early event in the antitumoral activity of this drug combination.

Interferon-beta can induce progesterone receptors in human endometrial adenocarcinoma.

The induction of estrogen and progesterone receptors (ER and PGR) has been reported in breast and endometrial cancer cells exposed to human fibroblast interferon-beta (hIFN-beta). Clinical verification of this finding might provide the rationale for new therapeutic approaches. This study was designed to evaluate whether clinical treatment with high doses of hIFN-beta induced ER and PGR in patients with endometrial adenocarcinoma. Two biopsies were obtained, 1 before and 1 after hIFN-beta treatment (3 x 10(6) i.m. every other day for 3 weeks) from 36 patients with endometrial adenocarcinoma. ER and PGR were determined with standard procedures using radiolabeled ligands. hIFN-beta treatment did not affect the proportion of ER-positive (i.e., >15 fmol/mg protein) or PGR-positive (i.e., >20 fmol/mg protein) cases. However, in patients with detectable ER and PGR at baseline, hIFN-beta raised the levels. Using a 35% difference before and after therapy as a cut-off, 72 and 79% of cases had increases in ER and PGR, respectively. The difference was highly significant for PGR. In patients with endometrial adenocarcinoma with undetectable ER or PGR, hIFN-beta did not induce the expression of these receptors. When the receptors were present they were upregulated by hIFN-beta. Whether this increase in receptor levels, particularly PGR, has therapeutic applications remains to be established.

Cell density-dependent modulation of basic fibroblast growth factor expression by human interferon-beta

The production of basic fibroblast growth factor (bFGF) by human renal cell carcinoma (HRCC) is associated with induction of angiogenesis. Incubation of HRCC cells with human interferon alpha (IFN-alpha) or interferon beta (IFN-beta) downregulates the expression of bFGF and, hence, angiogenesis. The purpose of this study was to analyze the downregulation of the expression of bFGF in HRCC cells by IFN-alpha and IFN-beta. Human HRCC SN12PM6 cells cultured under sparse conditions expressed 3-7-fold higher levels of steady-state bFGF-specific mRNA transcripts and cellular bFGF protein than did confluent cultures. IFN-alpha or IFN-beta inhibited the steady-state expression of bFGF mRNA transcripts and cellular bFGF protein in a concentration-dependent manner in sparse but not confluent cultures. Moreover, IFN-beta downregulated the transcription rate of bFGF genes and inhibited the de novo synthesis of bFGF protein only in sparse cultures. The results demonstrate that the inhibitory effects of IFN- and -beta on bFGF expression are cell-density dependent.

Mechanisms of transcriptional synergism of eukaryotic genes. The interferon-beta paradigm.

The virus-inducible enhancer of the human interferon-beta gene has served as an excellent example for the mechanisms controlling the activation and repression of transcription. This enhancer is activated by three different transcription factors that, with the help of the high mobility group protein HMG I(Y), assemble in a unique nucleoprotein complex that interacts as a unit with the basal transcriptional machinery. The assembly of unique enhancer complexes from similar sets of transcription factors may provide the specificity required for regulation of complex patterns of gene expression in higher eukaryotes.

A study of anterior pituitary hormones secretion in patients with glioma receiving interferon-beta treatment.

Human interferon beta (IFN-beta) has been used for the treatment of patients with benign and malignant astrocytomas. The effect of IFN-beta on pituitary function, however, has not been precisely evaluated before. In this study the serum levels of various anterior pituitary hormones including GH, PRL, ACTH, and TSH were measured to determine the effects of IFN-beta on pituitary endocrine function in 19 consecutive glioma patients receiving IFN-beta. Daily doses of 3 x 10(6) U of IFN-beta were administered as a 30-min intravenous drip infusion beginning at 0800 h every morning during the first week and then 4 times a week for additional 6 weeks. Blood samples were taken on the day prior to administration as controls (0900 h and 1500 h), and on the first day of administration (0900 h and 1500 h) and after 7 days of administration (0900 h) in order to determine the acute and chronic or integrated effects of IFN-beta on the above pituitary hormones. No significant change in serum concentrations of any of the pituitary hormones examined was observed, suggesting that human natural IFN-beta used for the treatment of gliomas has no significant effect on the secretion of these hormones from the pituitary in these patients.

Interleukin-8 gene expression by human dental pulp fibroblast in cultures stimulated with Prevotella intermedia lipopolysaccharide

Interleukin (IL)-8 mRNA expression was investigated in human dental pulp fibroblast cultures after stimulation with lipopolysaccharide (LPS) prepared from Prevotella intermedia and inflammatory cytokines. The expression of IL-8 mRNA and the release of IL-8 induced by P. intermedia LPS in pulpal fibroblast cultures were detected by Northern blot analysis and ELISA, respectively. The sufficient concentration of P. intermedia LPS on the IL-8 mRNA expression was 0.1 microgram/ml in pulpal fibroblast cultures. IL-8 mRNA levels began to increase after 2 h of exposure, reached a maximum at 4 to 8 h, and declined after 48 h, reaching the unstimulated level by 60 h. IL-8 production by the pulpal fibroblasts began to increase after 8 h of exposure upon stimulation with 10 microgram/ml of P. intermedia LPS. By contrast Salmonella LPS and synthetic lipid A did not increase IL-8 mRNA concentrations in pulpal fibroblast cultures. Recombinant human IL-1 alpha, beta, and tumor necrosis factor-alpha were capable of stimulating these cells to express IL-8 mRNA but natural human interferon-beta, gamma, and recombinant human IL-6 were incapable in our assay. These results suggest that pulpal fibroblasts are immunoresponsive cells and can elaborate IL-8 upon stimulation with P. intermedia LPS.

Scaffold/Matrix-Attached Regions: Structural Properties Creating Transcriptionally Active Loci

The expression characteristics of the human interferon-beta gene, as part of a long stretch of genomic DNA, led to the discovery of the putative domain bordering elements. The chromatin structure of these elements and their surroundings was determined during the process of gene activation and correlated with their postulated functions. It is shown that these "scaffold-attached regions" (S/MAR elements) have some characteristics in common with and others distinct from enhancers with which they cooperate in various ways. Our model of S/MAR function will focus on their properties of mediating topological changes within the respective domain.

Reconstitution of a High Affinity Binding Site for Type I Interferons

The type I interferon (IFN) receptor complex is assumed to be composed of multiple protein subunits. Recently, two proteins have been identified as potential receptor components, both of which share a high degree of structural homology with the immunoglobulin superfamily. One of these proteins, referred to as the human interferon alpha receptor (IFNAR), has been shown to be involved in interferon signal transduction, but it does not bind IFN with high affinity. A second putative receptor protein, named FLP40, has been cloned from human Daudi cells. Transfection of FLP40 into murine NIH 3T3 cells does not result in high affinity IFN binding. In this study, we demonstrate that when expressed in murine L929 cells neither IFNAR nor FLP40 by themselves are capable of binding human IFN-alpha 8. Co-expression of IFNAR and FLP40 results in cells capable of binding IFN-alpha 8 and IFN-alpha 2. Scatchard analysis of binding demonstrated the presence of high (KD 350 pM) and low (KD 4.0 nM) affinity binding sites. Binding of radiolabeled IFN-alpha 8 can be competed with either unlabeled IFN-alpha 8 or a recombinant form of human interferon beta, IFN-beta 1b, but not with IFN-gamma. Ligand binding of IFN-alpha 8 can be inhibited by antibodies directed against IFNAR providing further support for a role for this protein in the formation of a ligand binding site. This is the first demonstration indicating that two previously identified IFN receptor proteins, which individually do not bind type I IFN with high affinity, cooperate in the formation of a type I IFN receptor ligand binding complex.

Interferon beta mediated intracellular signalling traffic in human lymphocytes

The early molecular mechanisms activated by the treatment of human lymphocytes with human interferon beta have been studied. These identify an early increase with respect to control, in diacylglycerol (DG) levels as response to interferon treatment. Such a DG production was derived from the rapid and sequential activation of phosphoinositide specific phospholipase C and phospholipase D pathway. This suggests that a synergistic involvement of phosphatidylinositol-bis-phosphate (PIP 2) hydrolysis and phosphatidylcholine (PC) breakdown provide early molecular events upon the interaction between interferon beta and its cell surface receptors. This finally leads to the slowing down of cell growth.

Novel zinc chelators with dual activity in the inhibition of the kappa B site-binding proteins HIV-EP1 and NF-kappa B.

Both HIV-EP1 (also called PRDII-BF1 or MBP-1), a zinc finger protein, and NF-kappa B, a Rel family protein, bind to kappa B site present in the enhancer of multiple cellular and viral genes involved in immune function and inflammatory response including HIV-1 LTR and human interferon beta gene. When cells are exposed to extracellular stimuli such as virus or phorbol ester, the activity of both HIV-EP1 and NF-kappa B is induced. Thus, kappa B site-directed transcription could be regulated by two distinct proteins in a cooperative way. Novel heterocyclic compounds comprising (dimethylamino)pyridine and histidine units, i.e., 1-4, have been designed and synthesized, aiming at inhibition of these kappa B site-binding proteins to discriminate their functions. These compounds exhibited remarkable zinc-binding capability as revealed by NMR study. Compounds 1 and 2 showed a marked inhibitory effect on the DNA binding activity of HIV-EP1 by removing zinc without interfering with the DNA binding activity of NF-kappa B. Since it has been demonstrated that zinc somehow influences the DNA binding of NF-kappa B, the effect of these heterocyclic compounds and their zinc complexes on NF-kappa B was examined. Zinc complexes of 3 and 4 exhibited the inhibitory effect on the DNA binding of NF-kappa B and/or homodimeric complexes of p50 and p65 subunits of NF-kappa B without affecting HIV-EP1. Thus, it became possible to inhibit either one of the two kappa B site-binding proteins without inhibiting the other.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the proteins responsible for SAR DNA binding in nuclear matrix of Cucurbita pepo.

The nuclear matrices from White bush (Cucurbita pepo var. patisonina) cell nuclei have been isolated using three methods: I, standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; II, the same with pre-treatment of cell nuclei with 0.5 mM CuSO4 (stabilisation step); and III, method with extraction by lithium diiodosalicylate (LIS), and compared the polypeptide pattern. The isolated matrices specifically bind SAR DNA derived from human beta-interferon gene in the exogenous SAR binding assay and in the gel mobility shift assay. Using IgG against the 32 kDa endonuclease we have found in the DNA-protein blot assay that this protein is one of the proteins binding SAR DNA. We have identified three proteins with molecular mass of 65 kDa, 60 kDa and 32 kDa which are responsible for SAR DNA binding in the gel mobility shift assay experiments.

Growth inhibition of intracerebral rat glioma by transfection-induced human interferon-beta.

In our previous study on liposome-mediated transfection of the human interferon-beta (HuIFN-beta) gene into subcutaneously implanted human gliomas in nude mice, we found that HuIFN-beta was produced and secreted by the tumor cells and that the growth of solid tumors was completely inhibited. The present study investigated the growth-inhibitory effect of liposomes containing the HuIFN-beta gene inserted into a vector (pSV2IFN-beta) on T9 rat glioma implanted into the brains of rats. Tumor cells and liposomes containing pSV2IFN-beta or other additives were simultaneously injected into the brains of rats. HuIFN-beta was detected in solid gliomas growing in the brains of rats injected with liposomes and magnetic resonance imaging (MRI) showed that tumor growth was inhibited. In addition, the latent period until the appearance of neurological symptoms was significantly prolonged in rats treated with liposomes containing pSV2IFN-beta. However, the survival time of the treated rats was not significantly increased.

Interaction of the Pisum sativum nuclear matrix proteins with SAR DNA.

We have isolated the nuclear matrices from Pisum sativum cell nuclei using three methods: i. standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; ii. the same with pretreatment of cell nuclei with 0.5 mM CuSO4 (stabilisation step); and iii. method including lithium diiodosalicylate extraction. We compared the polypeptide pattern and residual DNA content of the nuclear matrices isolated. The nuclear matrices displayed a specific endonuclease activity which was due to the presence of a 32 kDa protein. The isolated nuclear matrices bound specifically the scaffold-attached (SAR) DNA derived from human beta interferon gene, in the exogenous SAR binding assay. Using the DNA-protein binding blot assay we demonstrated the presence of two nuclear matrix proteins of 66 kDa and 62 kDa which bound specifically SAR DNA.

Suppression of gelatinase production with decreased invasiveness of choriocarcinoma cells by human recombinant interferon beta

OBJECTIVE: Choriocarcinoma is a highly invasive gynecologic tumor, and hematogenous metastases frequently develop. To establish a molecular basis for antiinvasion therapy of choriocarcinoma, we examined the effects of human recombinant interferons on gelatinase production and invasion by choriocarcinoma cells. STUDY DESIGN: Using the five choriocarcinoma cell lines, we measured gelatinase activity by gelatin zymography. The effects of recombinant interferons (rIFN-α, riFN-β, and rIFN-γ) were then analyzed by Western blot analysis and chemoinvasion assay. RESULTS: High levels of 72 kd gelatinase activity were detected in the highly invasive choriocarcinoma cell lines, two of which also contained an active form of 72 kd gelatinase with an apoparent molecular mass of 88 kd. Gelatinase production was decreased by incubation with rIFN-β. In the chemoinvasion assay, only rIFN-β had an inhibitory effect on the invasiveness of tumor cells without a cytotoxic effect. CONCLUSION: Choriocarcinoma cells showed high 72 kd gelatinase activity, which suggested a role for the enzyme in vascular metastasis. Studies on the use of rIFN-β to inhibit metastasis of choriocarcinoma via suppressio of gelatinase production are warranted.

Differential effects of beta and gamma interferons on expression of major histocompatibility complex antigens and intercellular adhesion molecule‐1 in cultured fetal human astrocytes

We investigated the modulatory effects of human interferon beta (IFN-beta) and gamma (IFN-gamma) on expression of class I and II major histocompatibility complex (MHC) antigens and intercellular adhesion molecule-1 (ICAM-1) in fetal human astrocytes in culture using flow cytometry and immunocytochemistry. Under the baseline condition, class I MHC antigen and ICAM-1 were expressed in a moderate number (23 to 76%) of astrocytes, whereas class II MHC antigen was expressed in only a small number (0.3 to 8%) of astrocytes. Following a 72-hour treatment with IFN-gamma (10 to 100 U/ml), expression of all three antigens increased greatly. Expression of class I MHC antigen was also elevated by exposure to IFN-beta (10 and 100 IU/ml). However, IFN-beta did not significantly induce expression of class II MHC antigen or ICAM-1. Furthermore, IFN-beta significantly reduced IFN-gamma-induced expression of class II MHC antigen but not of class I MHC antigen or of ICAM-1. The differential effects of IFN-beta and IFN-gamma on expression of class I and II MHC antigens and ICAM-1 in fetal human astrocytes suggest that interferons serve as modulators of astrocyte function at sites of inflammation in the human CNS.

Carbohydrate‐Dependent Biological Activities of Glycosylated Human Interferon‐β on Human Hepatoblastoma Cells In Vitro

To evaluate the relationship between the sugar chain structure and biological activity, fibroblast-derived glycosylated human interferon-beta, Chinese hamster ovary cell-derived glycosylated recombinant human interferon-beta and Escherichia coli-derived unglycosylated recombinant human interferon-beta were evaluated using human hepatoblastoma cells in vitro. Native fibroblast interferon-beta expressed more cell-growth inhibitory action, 2'5'-oligoadenylate synthetase induction, and the inhibition of hepatitis B virus DNA replication than its asialoform and two recombinant interferon-betas. These results showed that the sugar chain structure of human interferon-beta affects its biological activity on human hepatoblastoma cells.

Human recombinant interferon beta in the treatment of relapsing-remitting multiple sclerosis

Human recombinant interferon beta in the treatment of relapsing-remitting multiple sclerosis

Identification of the rel family members required for virus induction of the human beta interferon gene.

We have carried out experiments to determine which members of the rel family of transcription factors are involved in virus induction of the beta interferon (IFN-beta) gene. First, we examined the inducibility of artificial DNA binding sites that preferentially interact with different homo- or heterodimeric combinations of rel proteins in vitro. We found that only those sites capable of binding the p50/p65 heterodimer are virus inducible. Second, we analyzed a series of mutant rel DNA-binding sites in the context of the intact IFN-beta promoter. We found a correlation between (i) sites capable of binding both the p50/p65 heterodimer and the high-mobility-group protein HMG I(Y) and (ii) virus inducibility. Third, cotransfection of the IFN-beta gene enhancer/promoter with plasmids capable of expressing several different rel proteins revealed that only the combination of p50 and p65 efficiently activated transcription. Finally, we have used antibodies directed against different rel proteins to show that virus-inducible protein-DNA complexes assembled on the IFN-beta enhancer in vitro contain both p50 and p65. We conclude that the p50/p65 heterodimer is responsible for the NF-kappa B-dependent activation of the IFN-beta gene promoter in response to virus infection.

Growth inhibition of human glioma cells by superinduced human interferon-beta.

Superinduction of human interferon-beta (HuIFN-beta) from human glioma cells has greater cytotoxicity than purified HuIFN-beta derived from fibroblasts. However, superinduction requires several reagents like polyI:polyC, cycloheximide, and actinomycin D, which may contaminate the conditioned medium and obscure the effect of superinduced HuIFN-beta. The present study used minimum doses of polyI:polyC and cycloheximide without actinomycin D to superinduce HuIFN-beta. The superinduced HuIFN-beta was purified by passing the medium through molecular sieve column chromatography. Fractionation of the eluate provided semipurified superinduced HuIFN-beta which demonstrated a growth inhibitory effect against both the U251-MG autologous human glioma cell line and the SK-MG-1 homologous glioma cell line. This effect was neutralized by addition of anti-HuIFN-beta monoclonal antibody (YSB-1). In a separate experiment, combinations of superinduction reagents were found not to have growth inhibitory effects because all inhibition in superinduced medium was completely neutralized by YSB-1. Superinduced HuIFN-beta has a pure growth inhibitory effect on both autologous and homologous glioma cells, so may affect autocrine secretion of cytokines.