Expression of the mouse c-abl type IV proto-oncogene product in the insect cell baculovirus system.

The cellular gene c-abl is the normal homologue of the transforming gene (v-abl) within the genome of the Abelson leukaemia virus. The cDNA sequence coding for the cellular form of the murine abl gene (c-abl type IV) has been inserted into the baculovirus transfer vector, pAc36C, so that the c-abl gene is under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells infected with the recombinant transfer vector in the presence of wild type AcNPV DNA yielded recombinant, polyhedrin negative virus that expressed moderate levels of the c-Abl protein (representing approx. 0.5-1% of the stained cellular proteins as determined by densitometric scanning). The insect derived c-Abl protein was compared to the P210-BCR/ABL protein from K562 cells, a cell line derived from a patient with chronic myelogenous leukaemia. Antibodies raised against synthetic peptides based on c-abl encoded peptides react with the insect derived c-Abl. In addition, the baculovirus derived c-Abl protein has a tyrosine kinase activity as demonstrated by phosphorylation of a synthetic polypeptide and also by autophosphorylation. Phosphoamino acid analysis of immunoprecipitated, autophosphorylated baculovirus derived c-Abl protein indicates that the majority of label incorporated is on the tyrosine residues. Immunofluorescence microscopy has been used to show that the majority of the c-Abl protein expressed in cells infected with recombinant virus is located in the nuclear and plasma membranes.

Relative transcriptional inducibility of the human interferon-alpha subtypes conferred by the virus-responsive enhancer sequence.

This paper addresses the role of transcriptional regulation in the determination of the levels of expression of different interferon-alpha subtypes secreted from Namalwa cells following infection with Sendai virus. Using RT-PCR to determine the relative abundance of mRNA species coding for the various subtypes, we found a general correlation with corresponding protein levels, indicative of a role for transcriptional control in the determination of levels of individual subtypes. We have used reporter gene constructs to compare the inducibility of the virus-response elements from the IFNA1, A2, A4, and A14 subtype genes cloned upstream of a secreted alkaline phosphatase gene. The inducibility of these reporter gene constructs broadly correlated with the relative mRNA abundances in both transiently and stably transfected Namalwa cells. During work with stable cell lines, we found that G418, the drug used for the selection of transfected cells, inhibited the induction of interferon by both Sendai virus and double-stranded RNA. This inhibition was reversible when G418 was removed from the medium 24 h before the addition of virus.

Detection of rare allelic variants of the interferon-alpha 2 gene in human genomic DNA.

We have analyzed human donor DNA for the presence of sequences corresponding to allelic variants of the IFN-alpha 2 locus. Using both restriction enzyme digestion of PCR-amplified fragments and sequence analysis of these fragments, we have identified the three reported allelic variants, IFN-alpha 2a, IFN-alpha 2b, and IFN-alpha 2c, in genomic DNA derived from donors of African or Afro-Caribbean origin. This is the first report of the IFN-alpha 2a and IFN-alpha 2c alleles occurring in human donor DNA and supports the view that these are variants of the predominant IFN-alpha 2b allele rather than arising from mutations occurring in cultured cells.

A regulatory domain within the virus-response element of the interferon alpha 1 gene acts as a transcriptional repressor sequence and determinant of cell-specific gene expression.

Type-I interferons are encoded by a multigene family, the major members of which are at least 13 IFN A subtypes and a single IFN B gene. IFNs A and B are induced in response to similar stimuli, such as virus infection and double-stranded RNA, but in different cell types: the induction of IFN A is almost exclusively restricted to cells of lymphoid origin, while IFN B has been found to be induced in a variety of cell types including fibroblasts. The virus-responsive enhancer element in the promoter region of IFN A family members is largely responsible for the differential expression of individual subtypes in responsive cells. In this paper we describe experiments which address the issue of the differential expression of IFN A and IFN B in different cell types. We show that IFN-beta is induced in a variety of cells of different origin, while not all of these are able to secrete IFN-alpha. By transfection of reporter gene constructs comprising the virus-responsive enhancer from the IFN A1 and IFN B genes, we show that this differential response is mediated at the level of transcription via these control elements. More detailed analysis of the function of these regions identifies specific sequences within the IFN A1 virus response element that has an inhibitory effect on expression in cells that are normally inducible, and is also implicated in the overall suppression of IFN A induction in non-inducible cells.

Inhibition of cell division by interferons: Changes in the transport and intracellular metabolism of thymidine in human lymphoblastoid (Daudi) cells.

The Daudi line of human lymphoblastoid cells shows a high sensitivity towards growth inhibition by human interferons. In cells pretreated with 70 reference units/ml of an interferon preparation for 48 h, the incorporation of exogenous [3H]thymidine into DNA is inhibited by as much as 85%. We are investigating the extent to which this effect reflects a true inhibition of the rate of DNA synthesis or whether it may be caused by changes in the metabolic utilization of exogenous thymidine by the cells. Interferon treatment results in a 30% inhibition of the rate of membrane transport and a 60% decrease in the rate of phosphorylation of [3H]thymidine in vivo. The latter effect is due to a decrease in V of thymidine kinase without any change in the value of Km for this enzyme. In addition to these changes, incorporation of [3H]uridine into DNA, which occurs as a result of the intracellular conversion of this precursor into thymidine nucleotides, is also inhibited by 75%, whereas RNA labelling by [3H]uridine is decreased by only 15% in interferon-treated cells. Thus several different metabolic events associated with thymidine nucleotide metabolism and DNA synthesis in Daudi cells are disrupted by interferon treatment.

Inhibition of cell division by interferons. The relationship between changes in utilization of thymidine for DNA synthesis and control of proliferation in Daudi cells.

Inhibition of the proliferation of Daudi cells by exposure to human lymphoblastoid interferons is associated with an early and marked decrease in the incorporation into DNA of exogenous [3H]thymidine when cells are incubated with trace amounts of this precursor. In contrast, incorporation of exogenous deoxyadenosine into DNA is unchanged under the same conditions. Interferon treatment results in a lowering of thymidine kinase activity, an effect which may be largely responsible for the inhibition of incorporation of labelled thymidine into DNA. At higher concentrations of exogenous thymidine, which minimize the contribution of intracellular sources to the dTTP pool, the inhibition of thymidine incorporation is abolished. Under conditions in which exogenous thymidine is rigorously excluded from the medium or, conversely, in which cells are entirely dependent on exogenous thymidine for growth, the magnitude of the inhibition of cell proliferation by interferons is the same as under normal culture conditions. We conclude that, even though cell growth is impaired, the rate of DNA synthesis is not grossly inhibited up to 48 h after commencement of interferon treatment. Furthermore, changes in neither the utilization of exogenous thymidine nor the synthesis of nucleotides de novo are responsible for the effect on cell proliferation.

Constitutive expression of a 2',5'-oligoadenylate synthetase cDNA results in increased antiviral activity and growth suppression.

The interferon (IFN)-induced enzyme 2',5'-oligoadenylate (2-5A) synthetase has been implicated in the development of antiviral activity in human and animal cells. However, its role in IFN-mediated growth inhibition remains unclear. To elucidate the function of 2-5A synthetase, we have stably introduced a human 2-5A synthetase cDNA into a human glioblastoma cell line (T98G). Constitutive expression of the cDNA in these cells is associated with increased levels of resistance to infection by encephalomyocarditis virus. One transfected subclone, which expresses elevated levels of 2-5A synthetase enzyme activity, also shows a reduced rate of cellular proliferation.

Highly efficient generation of recombinant baculoviruses by enzymatically medicated site-specific in vitro recombination.

We have used the Cre-lox system of bacteriophage P1 to develop a highly efficient in vitrosystem for construction of recombinant baculoviruses. A positive visual selection has been included to make identification of recombinant viral progeny rapid and straightforward. We report recombination frequencies as high as 5 x 10(7) recombinants/micrograms starting plasmid DNA and under certain conditions, up to 50% of the viral progeny are recombinants. Genes inserted into the baculovirus genome can be readily recovered in a simple one step process and re-inserted after manipulation if required. We have confirmed the structure of recovered plasmids by diagnostic restriction endonuclease digestion and the structure of recombinant viral genomes by Southern analysis. Possible uses and the significance of the system are discussed and experiments currently being done to improve it are described.

Inhibition of cell proliferation by interferons. 2. Changes in processing and stability of newly synthesized DNA in human lymphoblastoid (Daudi) cells.

The inhibition of proliferation of Daudi cells in culture by human interferons is characterized by a change in the kinetics of labelling of different size classes of newly synthesized DNA. Initially, labelled precursors are incorporated exclusively into small DNA (Okazaki fragments) in both control and interferon-treated cells, as revealed by alkaline sucrose gradient centrifugation. In the interferon-treated cells, there is enhanced labelling of this small DNA after short periods of incorporation and slower conversion to larger DNA size classes, in comparison with the DNA of control cells. This effect is apparent after 12 h of interferon treatment, coincident with the onset of the inhibition of cell proliferation. It becomes progressively more marked up to 4 days, by which time cell growth has ceased completely. Experiments using bromodeoxyuridine as a density label and analysis of radioactive DNA on caesium chloride/caesium sulphate gradients also reveal that some newly replicated DNA may be unstable and may turn over within a few hours of its synthesis. The label derived from DNA breakdown is efficiently reincorporated into newly synthesized molecules. It is suggested that interferon treatment inhibits DNA replication by activating DNA turnover rather than by directly inhibiting synthesis. This effect, together with the progressive retardation of conversion of Okazaki fragments to larger DNA, may lead to the eventual failure of cell proliferation.

The effect of interferon on cells deficient in nucleoside transport or lacking thymidine kinase activity.

Mutants of mouse T-lymphoma S49 cells lacking thymidine kinase activity or deficient in nucleoside transport were selected by growth in the presence of 5'-fluorodeoxyuridine and their sensitivity to interferon tested. All five thymidine kinase and both transport deficient mutants were sensitive to the antiproliferative effects of interferon. The replication of encephalomyocarditis virus was also inhibited by interferon, and the intracellular levels of (2'-5')oligo-adenylate synthetase were elevated in all mutants tested. These results suggest that an intact nucleoside transport system or thymidine kinase activity are not essential for the expression of interferon sensitivity in these cells.

Characterization and regulation of alpha-interferon receptor expression in interferon-sensitive and -resistant human lymphoblastoid cells.

Interferon-sensitive (IFN-S) and IFN-resistant (IFN-R) Daudi lymphoblastoid cells were studied for IFN-alpha receptor expression and regulation by steady state and kinetic procedures, utilizing a homogeneous 125I-IFN-alpha 2 probe. Heterogeneity in the binding of this probe to IFN-S cells was determined to result from negatively cooperative interactions between an initially homogeneous class of IFN receptor. No such heterogeneity was noted in the IFN-R cells, indicating an apparent difference in the interaction of IFN-alpha 2 with these cells. The apparent dissociation constants (Kd) for IFN-S cell receptors were calculated to be 1 X 10(-10) M and 1 X 10(-8) M, for the high and low affinity sites, respectively. The Kd for sites on the IFN-R cells was estimated to be 4 X 10(-9) M. IFN-R and IFN-S cells expressed 2.4 X 10(4) and 3.5 X 10(4) binding sites per cell, respectively, representing an increase of at least 6-fold over previous reports of IFN-S Daudi IFN receptor density. Both IFN-S and IFN-R cells were capable of down-regulating expression of the IFN-alpha receptor in response to low concentrations of IFN-alpha 2. Furthermore, both cell lines were shown to be capable of internalizing specifically bound 125I-IFN-alpha 2 to an equivalent degree. Accordingly, we propose that the relative insensitivity of the Daudi IFN-R phenotype involves the loss of a high affinity interaction between cellular receptors and IFN-alpha 2, in addition to the reduced level of expressed low affinity binding sites.

Reversal of the double-stranded-RNA-induced inhibition of protein synthesis by a catalytically inactive mutant of the protein kinase PKR.

The interferon-inducible double-stranded-RNA(dsRNA)-dependent protein kinase PKR has been implicated in both the antiviral and cell growth-regulatory effects of the interferons. Over-expression of the wild-type form of this protein inhibits cell proliferation, whereas over-expression of inactive mutant forms transforms cells to a tumourigenic phenotype. It has been suggested that mutant PKR exerts a dominant negative effect on the activity of the wild-type protein kinase. We have investigated this possibility using the rabbit reticulocyte cell-free translation system in which protein synthesis is inhibited by dsRNA due to activation of PKR and phosphorylation of initiation factor eIF-2. Addition of a highly purified inactive PKR mutant, synthesised in a baculovirus-infected insect cell system, rescues protein synthesis from inhibition by low concentrations of dsRNA in a dose-dependent manner. The PKR mutant has no effect on protein synthesis in the absence of dsRNA or in the presence of another inhibitory protein kinase, the haem-controlled repressor. Inhibition of translation can be re-established in the presence of the mutant PKR by adding a higher concentration of dsRNA. These results suggest that inactive mutant PKR does exert a dominant negative effect on wild-type PKR and that this may be due to competition for dsRNA binding.

Regulation of the interferon-inducible protein kinase PKR and (2'-5')oligo(adenylate) synthetase by a catalytically inactive PKR mutant through competition for double-stranded RNA binding.

The interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a tumour suppressor gene product. Catalytically inactive mutants of PKR give rise to a tumorigenic phenotype when overexpressed in NIH-3T3 fibroblasts and this has been attributed to a dominant negative effect on the activity of the wild-type enzyme. Here we show that the mutant with Lys296 replaced by Arg, [K296R]PKR, not only inhibits the protein kinase activity of wild-type PKR but is also inhibitory towards another double-stranded RNA-dependent enzyme, the 40-kDa form of (2'-5')oligo(adenylate) synthetase. Inhibition of both wild-type PKR and (2'-5')oligo(adenylate) synthetase is reversed by adding higher concentrations of double-stranded RNA. These results suggest competition between [K296R]PKR and wild-type PKR or (2'-5')oligo(adenylate) synthetase for limiting amounts of double-stranded RNA. Moreover, the data imply that the tumorigenic effect of this PKR mutant could be due to inhibition of additional pathways requiring low levels of double-stranded RNA for activation and cannot be unambiguously attributed to inhibition of endogenous PKR itself.

Characterization and expression of a murine gene homologous to human EPA/TIMP: a virus-induced gene in the mouse.

A genomic clone encompassing the entire coding region of a murine gene homologous to human erythroid potentiating activity/tissue inhibitor of metalloproteinase (EPA/TIMP) was isolated and sequenced. Based on alignment with human EPA/TIMP cDNAs we deduce a structure comprising five exons and four introns extending over 4.3 kb of DNA. In mouse and hamster cell lines transcription from this gene and interferon genes is induced by Newcastle Disease virus (NDV). Examination of the 5'-flanking sequences of the gene reveals a set of repeated elements with structural similarity to those previously described as inducer-responsive elements in the human IFN-beta 1 gene. The 4.3-kb DNA fragment encompassing the homologous murine EPA/TIMP gene was transfected into human T98G cells and transfectants tested for NDV inducibility. In contrast to the endogenous human gene, the integrated murine EPA/TIMP gene was NDV-inducible and TIMP activity was detectable in the cell culture fluid.

Relationship of cellular oncogene expression to inhibition of growth and induction of differentiation of Daudi cells by interferons or TPA.

Human alpha or beta interferons inhibit the proliferation of Daudi Burkitt lymphoma cells and induce the differentiation of these cells towards a mature plasma cell phenotype. Similar responses are seen when Daudi cells are treated with the phorbol ester, TPA. Both interferons and TPA down-regulate expression of the c-myc oncogene in these cells. Although TPA can mimic the effect of interferon on cell differentiation, it does not induce 2'5' oligoadenylate synthetase or the interferon-sensitive mRNAs, 6-16 or 9-27. Thus chronic stimulation of protein kinase C by TPA cannot mimic all of the effects of interferon treatment on gene expression. Inhibition of ADP-ribosyl transferase activity by 3-methoxybenzamide impairs interferon- or TPA-induced differentiation of Daudi cells. This agent induces a higher level of c-myc mRNA in the cells and stimulates the incorporation of [3H]thymidine into DNA; although these effects are partially counteracted by interferon or TPA treatment, the elevated expression of the c-myc gene may be sufficient to prevent terminal differentiation and allow cell proliferation to continue.

Molecular cloning of two new interferon-induced, highly related nuclear phosphoproteins.

During the molecular cloning of the human dsRNA activated-p68 kinase (PKR), polyclonal antibodies against PKR selected, in addition to cDNAs corresponding to PKR, another cDNA presenting only slight homology with PKR cDNA. This cDNA recognized an mRNA species of 2 kilobases induced by both alpha- and gamma-interferons. Its transcription did not require protein synthesis. On further library screening, it selected two highly related cDNAs, referred to as 75 and 41, displaying perfect homology over 612 base pairs and divergent at both ends. In addition, cDNA 75 presents an insertion of 150 base pairs highly homologous to a region common to both sequences. The 75 and 41 peptidic sequences are very hydrophilic, rich in basic amino acid residues, and contain several potential phosphorylation sites for different serine/threonine kinases. Furthermore, they present two protamine- and histone-like nuclear targeting sequences as well as some homology with helix-loop-helix motifs of some DNA-binding proteins. The 75-encoded product, which resolved as a 52-kDa protein after in vitro expression in rabbit reticulocyte lysates, was found to migrate as a 65-67-kDa protein after in vivo expression in insect cells. In accord with sequence data, this 65-67-kDa protein was found to be phosphorylated in vivo in the insect cells and was recovered from the membrane/nuclear pellet. In contrast, the 41-encoded product (30-kDa protein in reticulocyte lysates) could not be expressed in vivo, as it provoked a rapid and severe shut-off of protein synthesis in insect cells. The function of the 75 and 41 proteins and their relation to PKR remains to be determined. However, the presence of nuclear targeting sequences, phosphorylation sites, and helix-loop-helix motif is consistent with a role of these proteins in the mechanism of transduction of the interferon action.

Inhibition of cell proliferation by interferons. 1. Effects on cell division and DNA synthesis in human lymphoblastoid (Daudi) cells.

Treatment of Daudi cells with human lymphoblastoid interferons for up to 5 days progressively inhibits cell proliferation. For the first 3 days cells continue to grow but with prolonged doubling times; subsequently, net proliferation ceases and is accompanied by a loss of cell viability. We have investigated the changes in labelling of DNA with radioactive precursors which occur during the first phase of the response to interferon treatment. We have shown previously [Gewert et al. (1981) Eur. J. Biochem. 116, 487-492] that inhibition of incorporation of [3H]thymidine into DNA can be accounted for by impairment of thymidine transport and thymidine kinase activity. In spite of this inhibition, the total intracellular dTTP pool is larger in interferon-treated than in control cells. Because of these changes it has been necessary to use other methods to determine whether interferon treatment inhibits the overall rate of DNA synthesis. The results of experiments employing (a) moderately high thymidine concentrations or (b) incorporation of radioactivity from deoxynucleoside triphosphates into DNA in detergent-lysed or permeabilised cell systems indicate that there is in fact relatively little inhibition of the overall rate of DNA synthesis in cells exposed to up to 100 units/ml of interferons for at least 48 h. Furthermore, a similar proportion of cells incorporate [3H]thymidine in control and interferon-treated cultures and there is only a small decrease in the number of cells in S phase after interferon treatment, as revealed by fluorescence-activated cell sorting. These results indicate that cell proliferation may be regulated in this system by a mechanism in which there is a loss of coordination between the initiation of DNA synthesis and the subsequent events required for cell division.

Human 2-5A synthetase: characterization of a novel cDNA and corresponding gene structure.

The enzyme 2-5A synthetase is induced in cultured cells in response to interferon (IFN) treatment. A lambda gt10 cDNA library of mRNA from IFN-induced Daudi lymphoblastoid cells was screened with oligonucleotide probes. Several overlapping cDNAs were isolated and shown to be derived from the human synthetase gene using filter selection and oocyte microinjection assays. The nucleotide sequence of one of these, cDNA 8-2, extended the 2-5A synthetase sequence already described 72 bp in the 5' direction but was found to differ significantly in coding sequence at the 3' end. The longest cDNA isolated (6-2) was approximately 1.4 kb. By Northern hybridization analysis single mRNAs of 1.7 kb were detected in Daudi and T98G (glioblastoma) cells. However, in HeLa cells, four mRNAs ranging in size from 1.5 to 3.5 kb were found, one of which differed at the 3' end. Analysis of both phage and cosmid genomic clones and comparison with genomic DNA indicate that there is a single gene for 2-5A synthetase, comprising at least six exons and five introns, which can undergo a novel form of alternative RNA processing depending on cell type.

Activation of the interferon-inducible (2'-5') oligoadenylate synthetase by the Epstein-Barr virus RNA, EBER-1.

The 2'-5' oligoadenylate synthetases and the protein kinase PKR are both interferon-induced, double-stranded RNA-dependent proteins that play important roles in the antiviral effects of the interferons and in cellular growth control. Both enzymes are activated by natural or synthetic dsRNAs and by single-stranded RNAs that possess extensive secondary structure. This report describes the effects of the small Epstein-Barr virus-encoded RNA EBER-1 on the regulation of 2-5(A) synthetase activity. We demonstrate that EBER-1 RNA binds to and activates the human 40-kDa 2-5(A) synthetase in a dose-dependent manner. The efficiency of EBER-1 as an activator of 2-5(A) synthetase is approximately 25% of that of the synthetic double-stranded RNA poly(I)/poly(C), and poly(I)/poly(C) further stimulates enzyme activity even in the presence of a high concentration of EBER-1. Conversely, EBER-1 neither stimulates nor inhibits 2-5(A) synthetase that has been activated by a high concentration of poly(I)/poly(C). Competitive binding assays suggest that the relative affinity of the enzyme for poly(I)/poly(C) is considerably higher than that for EBER-1. Our data indicate that EBER-1, like VAI RNA of adenovirus, TAR RNA of HIV-1, and Rex-RE RNA of HTLV-1, is able to activate the 2-5(A) synthetases. The significance of why several viruses may activate the 2-5(A) synthetase/RNase L-mediated RNA degradation pathway is discussed.

Differential binding of human interferon-alpha subtypes to receptors on lymphoblastoid cells.

Highly purified human interferon-alpha subtype A (HuIFN alpha A) was iodinated for use in direct ligand binding studies on human lymphoblastoid (Daudi) cells. Unlabelled preparations of HuIFN alpha subtypes A, C, D, and hybrid molecules AD (Bgl II), AD (Pvu II), and DA (Bgl II) showed different responses in competition experiments with labelled alpha A probe. Specifically, IFNs alpha D and alpha DA were unable to displace the probe, whereas IFNS alpha A, alpha C, and the hybrid alpha ADs showed similar competition curves. These results support a two-idiotope model of IFN recognition by its receptor. IFN effects on [3H]-thymidine incorporation and cell growth (long term effects) did not reflect the apparent affinities of HuIFN alpha subtypes for cell surface receptor.