Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth.

Interferon-inducible membrane proteins of approximately 17 kDa have been suggested to play a role in the antiproliferative activity of interferons based on (1) their pattern of induction in interferon-sensitive and -resistant cell lines and (2) the ability of a membrane fraction enriched in 17-kDa proteins to inhibit cell growth. To gain insight into the nature of the proteins that mediate the antiproliferative activity of interferons, a monoclonal antibody, 13A5, was generated that reacted specifically with a 17-kDa interferon-inducible cell surface protein. The expression pattern of this 17-kDa protein by human cell lines correlated with sensitivity to the antiproliferative activity of interferons. To obtain information regarding the structure of this protein, the 13A5 antibody was used to screen COS cells transfected with a human cDNA expression library. Sequence analysis of a full-length cDNA clone revealed identity with the 9-27 cDNA, previously isolated on the basis of its interferon inducibility by differential screening. In addition, the 17-kDa protein encoded by the 9-27 gene was shown to be identical to the Leu-13 antigen. Leu-13 was previously identified as a 16-kDa interferon-inducible protein in leukocytes and endothelial cells and is a component of a multimeric complex involved in the transduction of antiproliferative and homotypic adhesion signals. These results suggest a novel level of cellular regulation by interferons involving a membrane protein, encoded by the interferon-inducible 9-27 gene, which associates with other proteins at the cell surface, forming a complex relaying growth inhibitory and aggregation signals.

Regulation of gene expression by cytokines and virus in human cells lacking the type-I interferon locus.

A number of genes that are induced by type-I interferons are also activated by one or more other inducers, including double-stranded RNA, viruses, interferon-gamma, interleukin-1 and tumor necrosis factor. However, these inducers can also activate the expression of type-I interferons. Thus, the activation of type-I interferon-inducible genes by these other inducers could be direct, or a secondary consequence of the induction of interferon. To distinguish between these possibilities, we have used cell lines lacking all type-I interferon genes to study the direct effect of potential inducers on the expression of 14 interferon-inducible human genes. We show that double-stranded RNA, virus, interferon-gamma or tumor necrosis factor-alpha can act directly to induce specific subsets of type-I interferon-inducible genes in the absence of any possible type-I interferon involvement. The cis-acting element which confers inducibility by type-I interferon has been shown in some cases to confer inducibility by interferon-gamma, double-stranded RNA or virus as well. However, not all promoters containing such an element respond to both interferon and other inducers. Thus, the ability of a given gene to respond to different inducers most likely depends on the exact nature and specific combination of cis-acting elements present in its promoter.

CD69 is expressed on Daudi cells in response to interferon-alpha.

An approach to obtain monoclonal antibodies directed against cell surface proteins induced by interferon has been developed in order to characterize such proteins and determine their role. Hybridomas obtained by fusion of murine myeloma cells and spleen cells of mice immunized with interferon-alpha-treated Daudi cells were screened for the production of antibodies reacting differentially with interferon-alpha-treated and untreated Daudi cells. One such hybridoma, 2D5, produced an antibody reacting with a 28/32 kDa homodimeric protein (p28/32) expressed at the surface of Daudi cells in response to IFN-alpha treatment. IFN-alpha treatment also increased the basal level of p28/32 detected on peripheral blood leukocytes (PBL). 2D5 Antibody was used to probe the expression of p28/32 on different cells and in response to various inducers. It appears that 2D5 reacted in fact with CD69, a marker of leukocyte activation and that, following IFN-alpha treatment, CD69 was not induced on all cultured cell lines tested. Interestingly, IFN-gamma was also able to induce CD69 expression on a restricted number of cell lines but the induction pattern only partially overlapped that of IFN-alpha. As expected, activation of cells with phorbol myristate acetate (PMA) resulted in a notable increase in the level of CD69 on all cell lines considered except for the epithelial and fibroblastic types.

Post-transcriptional effect of ultraviolet light on gene expression in human cells. Stabilization of cytokine-induced and poly(I).poly(C)-induced messenger RNA.

It is well established that ultraviolet light modulates gene expression in mammalian cells, particularly at transcriptional and post-translational levels. The present study was undertaken to investigate whether the fate of mRNA is also altered in ultraviolet-light-irradiated human cells. In order to facilitate distinction between transcriptional and post-transcriptional effects, this analysis has focused on six genes whose transcription is conditional on the supply of exogenous inducers, interferon-alpha, interleukin-1 alpha or the double-stranded RNA, poly(I).poly(C). Human cells induced to express these genes were found to retain a significantly higher concentration of corresponding transcripts when irradiated with ultraviolet light at the end of the inducing treatment. This stimulation was due to dose-dependent ultraviolet-light stabilization of preformed mRNA, as shown by run-on and pulse/chase experiments. This work uncovers a new facet of the cellular response to genotoxic stresses, i.e. extension of the life-span of transcription products. Whether this stabilizing effect contributes to cell recovery by promoting gene expression remains to be determined.

Human thymosin-beta 4/6-26 gene is part of a multigene family composed of seven members located on seven different chromosomes.

We have isolated a cDNA encoding the human interferon-inducible gene 6-26, by screening a cDNA library with an oligodeoxynucleotide probe. Its sequence was found to be identical to that of the human thymosin-beta 4 cDNA, which encodes a protein present in most cell types, but whose function is not clear at present. By hybridization of the thymosin-beta 4/6-26 cDNA to the DNA of a panel of human-rodent somatic cell hybrids, we found that at least seven genes homologous to this cDNA are present in the human genome. We localized these genes, some of which might be pseudogenes, to seven distinct chromosomes, namely, chromosomes 1, 2, 4, 9, 11, 20, and X.

Analysis of interferon-inducible genes in cells of chronic myeloid leukemia patients responsive or resistant to an interferon-alpha treatment.

Recombinant human interferon-alpha (IFN-alpha) can induce a hematologic remission in patients with chronic myeloid leukemia. However, some patients are resistant and others develop late resistance to the IFN-alpha treatment. To understand the molecular mechanism of this resistance, we have analyzed the expression of 10 IFN-inducible genes in the cells of three resistant patients, two responsive patients, and six healthy controls. Northern blot hybridizations showed that all the genes were induced in in vitro IFN-alpha treated peripheral blood cells of the patients and healthy controls. These genes were also inducible in peripheral blood and bone marrow cells of two out of two resistant patients administered an injection of IFN-alpha. We conclude that the resistance to the IFN-alpha treatment of the chronic myeloid leukemia patients we studied is not due to (1) the absence of induction of any of the 10 IFN-inducible genes we studied, including the low-molecular-weight 2'-5'oligoadenylate synthetase; (2) the presence of an antagonist of IFN-alpha in the peripheral blood or bone marrow cells; and (3) the presence of neutralizing anti-IFN-alpha antibodies.

Chromosomal localization of two human genes inducible by interferons, double-stranded RNA, and viruses.

Human IFI-15K and 6-16 genes are transcriptionally stimulated by interferons, double-stranded RNA, and viruses. By screening a cDNA library with oligodeoxynucleotide probes, we have isolated complete copies corresponding to these two genes. These cDNA clones allowed us to localize the IFI-15K and 6-16 genes on human chromosome 1 by somatic cell hybridization.

2-Aminopurine selectively blocks the transcriptional activation of cellular genes by virus, double-stranded RNA and interferons in human cells.

Interferons, double-stranded RNA and viruses induce the transcription of partly overlapping sets of cellular genes. We have studied the regulation of 11 interferon-inducible genes by these agents and found that four of them were also directly inducible by virus and double-stranded RNA, and two by virus only. We have investigated whether an inhibitor of interferon-beta gene activation, 2-aminopurine, would block this induction process. Induction of these genes by virus and double-stranded RNA was indeed blocked by 2-aminopurine. Since a single cis-acting element can confer inducibility both to interferons, and to virus and double-stranded RNA, we tested the effect of 2-aminopurine on gene activation by interferon-alpha and interferon-gamma. Remarkably, in all the cell lines tested, these induction processes and the establishment of an antiviral state were blocked by the drug. These observations contrast with previous reports. The inhibitory effect of this drug on gene induction was exerted in a selective fashion and at the transcriptional level. This indicate that for the virus-, double-stranded-RNA-, and interferons-mediated gene induction, an early and similar step in signal transduction was affected by 2-aminopurine.

Identification of a translation inhibitory element (TIE) in the 3' untranslated region of the human interferon-beta mRNA.

We have previously reported that the 3' untranslated region (UTR) of the human interferon-beta mRNA has an inhibitory effect on the mRNA translation both in vitro, in a rabbit reticulocyte lysate, and in vivo, in the Xenopus oocyte. In the present study, we identify the sequence in the 3' UTR which is responsible for this translation inhibition. We show that this sequence is located between the 100th and 161st nucleotides downstream from the translation stop codon. It contains several repeats of the A + U-rich consensus octanucleotide UUAUUUAU, which is also present in the 3' UTR of several mRNAs involved in the inflammatory response. We also demonstrate here that the inhibitory effect of the sequence on the mRNA translation does not depend on its position in relation to the termination codon. However, no inhibition of translation is observed when this sequence is inserted in the 5' UTR of the mRNA. The removal of the translation inhibitory sequence not only improves the mRNA translation in Xenopus oocytes but it also strongly decreases the IFN-beta mRNA stability in those cells. This suggests that, in this system at least, the mRNA degradation is linked to its translational efficiency.

Regulation of two interferon-inducible human genes by interferon, poly(rI).poly(rC) and viruses.

The IFI-56K and IFI-54K genes are transcriptionally stimulated when cells are treated by interferon. We have previously shown that the IFI-56K gene is in addition directly induced by poly(rI).poly(rC), and inducer of interferon-beta. Since the regulation of the IFI-56K and IFI-54K genes by interferon are very much alike, we tested whether the IFI-54K gene is also directly regulated by poly(rI).poly(rC). Treatment of various cell lines with poly(rI).poly(rC) leads to a clear accumulation of the IFI-54K mRNA to a level which sometimes even exceeds that obtained with high doses of interferon. Several interferon-resistant cell lines were investigated for the inducibility of both the IFI-56K and IFI-54K genes by interferons, poly(rI).poly(rC) and viruses (which are the natural inducers of interferon-alpha and -beta). Both genes appear to be coordinately regulated by these inducers. It was thus interesting to search for common regulatory element(s) in the control region of these two genes. The IFI-54K gene promoter region was isolated, from which a 520-base-pair segment was sequenced and compared with the promoter region of the IFI-56K gene that we had previously sequenced. The only homology was found is a well conserved 19-bp segment located just upstream of the TATA box of these genes; interestingly, this sequence is also homologous to the minimal region needed for the inducibility by poly(rI).poly(rC) of the interferon-beta gene. This conserved sequence might be responsible for the coordinate induction of the IFI-56K and IFI-54K genes by interferon, poly(rI).poly(rC) and viruses.

The IFI-56K and IFI-54K interferon-inducible human genes belong to the same gene family.

The IFI-56K and IFI-54K human genes are coordinately regulated by interferon, double-stranded RNA and viruses in a number of cell lines. These genes encode polypeptides of 56 and 54 kDa, respectively, whose function remains to be determined. We analysed the possible structural relatedness between these syntenic and similarly regulated genes. We found that they are very closely related at the protein, mRNA and promoter levels. This suggests that the IFI-56K and IFI-54K genes are members of a gene family, which probably arose from duplication of an ancestor gene.

New inducers revealed by the promoter sequence analysis of two interferon-activated human genes.

In order to investigate the molecular basis of the regulation of interferon-inducible genes, we isolated the promoter region of two such genes coding for the (2'-5')oligo(adenylate) synthetase and a 56-kDa protein (IFI-56K). The regions surrounding the cap site were sequenced and compared with the sequences of vertebrate and viral DNA present in the Genbank data bank. Small DNA segments were found in both genes which are homologous to part of the promoter region of other genes, such as those of interferon-beta, tumor necrosis factor beta, interleukin-2 and its receptor. Since these homologies were found located in functionally important regions of these genes, we tested whether their inducers also enhance the (2'-5')oligo(adenylate) synthetase and IFI-56K gene expression. We found that poly(rI).poly(rC) and interleukin-1, activators of the interferon-beta gene and of T lymphocytes respectively, are both able to enhance IFI-56K mRNA accumulation in all cell lines tested. Cycloheximide even superinduces this gene when added together with poly(rI).poly(rC) and interleukin-1 (but not when added with interferon). We showed that these inductions are direct and not mediated by interferon produced by cells in response to poly(rI).poly(rC) or interleukin-1. The promoter sequence analyses have thus led to the discovery of unexpected inducers, i.e. an interferon inducer such as poly(rI).poly(rC) is also able to directly induce a gene that is under the control of interferon.