Exogenous administration of protease-resistant, non-matrix-binding IGFBP-2 inhibits tumour growth in a murine model of breast cancer.

BACKGROUND: Insulin-like growth factors (IGF-I and IGF-II) signal via the type 1 IGF receptor (IGF-1R) and IGF-II also activates the insulin receptor isoform A (IR-A). Signalling via both receptors promotes tumour growth, survival and metastasis. In some instances IGF-II action via the IR-A also promotes resistance to anti-IGF-1R inhibitors. This study assessed the efficacy of two novel modified IGF-binding protein-2 (IGFBP-2) proteins that were designed to sequester both IGFs. The two modified IGFBP-2 proteins were either protease resistant alone or also lacked the ability to bind extracellular matrix (ECM). METHODS: The modified IGFBP-2 proteins were tested in vitro for their abilities to inhibit cancer cell proliferation and in vivo to inhibit MCF-7 breast tumour xenograft growth. RESULTS: Both mutants retained low nanomolar affinity for IGF-I and IGF-II (0.8-2.1-fold lower than IGFBP-2) and inhibited cancer cell proliferation in vitro. However, the combined protease resistant, non-matrix-binding mutant was more effective in inhibiting MCF-7 tumour xenograft growth and led to inhibition of angiogenesis. CONCLUSIONS: By removing protease cleavage and matrix-binding sites, modified IGFBP-2 was effective in inhibiting tumour growth and reducing tumour angiogenesis.

Detailed mapping of the ERG-ETS2 interval of human chromosome 21 and comparison with the region of conserved synteny on mouse chromosome 16.

We have carried out a detailed annotation of 550 kb of genomic DNA on human chromosome 21 containing the ERG and ETS2 genes. Comparative genomic analysis between this region and the interval of conserved synteny on mouse chromosome 16 indicated that the order and orientation of the ERG and ETS2 genes were conserved and revealed several regions containing potential conserved noncoding sequences. Four pseudogenes including those for small protein G, laminin receptor, human transposase protein and meningioma-expressed antigen were identified. A potentially novel gene (C21orf24) with alternative mRNA transcripts, consensus splice donor and acceptor sites, but no coding potential nor murine orthologue, was identified and found to be expressed in a range of human cell lines. We have identified four novel splice variants that arise from a previously undescribed 5' exon of the human ERG gene. Comparison of the cDNA sequences enabled us to determine the complete exon-intron structure of the ERG gene. We have also identified the presence of noncoding RNAs in the first and second introns of the ETS2 gene. Our studies have important implications for Down syndrome as this region contains multiple mRNA transcripts, both coding and potentially noncoding, that may play as yet undescribed roles in the pathogenesis of this disorder.

Generation and characterization of recombinant unmodified and phosphorylatable murine IFN-alpha1 in the methylotropic yeast Pichia pastoris.

In order to generate reagents to study the murine type I interferon (IFN) system, recombinant murine IFN-alpha1 (rMuIFN-alpha1) protein was expressed in the methylotropic yeast Pichia pastoris. rMuIFN-alpha1 with a phosphate acceptor site engineered at the C-terminus (rMuIFN-alpha1P) to enable radiolabeling by gamma(32)P-ATP and cAMP-dependent protein kinase was also generated. Proteins of 20, 25 (MuIFN-alpha1) and 25.5 (MuIFN-alpha1P), kDa were detected in the yeast growth medium, had type I IFN activity, and were recognized by antimurine L929 cell IFN antibodies. The MuIFN-alpha1 proteins produced in P. pastoris were a mixture of glycosylated and unglycosylated forms, with sugars of approximately 5 kDa added via N-linked glycosylation. The recombinant proteins were highly purified using a single RP-HPLC elution step, and their authenticity was confirmed by amino-terminal amino acid sequencing. The MuIFN-alpha1 and MuIFN-alpha1P protein preparations had specific antiviral activities of 1.3 x 10(7) and 4.7 x 10(6) IU/mg protein, respectively. MuIFN-alpha1P could be radiolabeled to a high specific radioactivity (0.6-2 x 10(8) cpm/microg protein) with gamma(32)P-ATP and cAMP-dependent protein kinase without significantly altering its biologic activity or electrophoretic properties. Binding experiments on COS-7 cells transiently transfected with MuIFNAR-2 and IFNAR-2 demonstrated specific and dose-dependent binding of gamma(32)P-ATP-MuIFN-alpha1P to cell surface type I IFN receptors.

Identification and characterization of polymorphisms at the HAS alpha1-acid glycoprotein (ORM*) gene locus in Caucasians

Human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is a major acute phase protein that is thought to play a crucial role in maintaining homeostasis. Human AGP is the product of a cluster of at least two adjacent genes located on HSA chromosome 9. Using a range of restriction endonucleases we have investigated DNA variation at the locus encoding the AGP genes in a group of healthy Caucasians. Polymorphisms were identified using BamHI, EcoRI, BglII, PvuII, HindIII, TaqI and MspI. Nonrandom associations were found between the BamHI, EcoRI and BglII RFLPs. The RFLPs detected with PvuII, TaqI and MspI were all located in exon 6 of both AGP genes. The duplication of an AGP gene was observed in 11 of the individuals studied and was in linkage disequilibrium with the TaqI RFLP. The identification and characterization of these polymorphisms should prove useful for other population and forensic studies

A novel member of the STOMATIN/EPB72/mec-2 family, stomatin-like 2 (STOML2), is ubiquitously expressed and localizes to HSA chromosome 9p13.1.

A cDNA encoding a novel second member of the Band7/stomatin-like/SPFH domain family in humans designated stomatin-like 2 (STOML2) has been isolated using the technique of cDNA Representational Difference Analysis. The STOML2 cDNA encoded a 356 amino acid residue polypeptide with a predicted molecular weight of 38.5 kDa. The predicted polypeptide sequence of STOML2 could be delineated into three major domains: an N-terminal alpha-helical region; a domain with significant similarity to a 172 amino acid region of the HSA stomatin polypeptide, composed of an alternating alpha-helical and beta-sheet structure and a C-terminal domain that was mostly alpha-helical. The stomatin-like domain was observed in 51 other proteins with potentially diverse functions. Based on its homology to stomatin, STOML2 was predicted to be cytoplasmically located. However, unlike most of the other proteins containing stomatin-like domains, the predicted STOML2 polypeptide does not contain a transmembrane region although the presence of N-myristoylation sites suggest that it has the potential to be membrane-associated. Northern blot analysis of a panel of poly(A)(+) mRNA from normal human adult tissues showed that a single 1.3-kb mRNA transcript encoding STOML2 was ubiquitously expressed, with relatively higher levels in skeletal muscle and heart compared to other tissues. Comparison of the STOML2 cDNA sequence with human genomic DNA indicated that the gene encoding STOML2 was 3,250 bp long and consisted of ten exons interrupted by nine introns. We have mapped STOML2 to HSA chromosome 9p13.1, a region that is rearranged in some cancers and thought to contain the gene responsible for acromesomelic dysplasia.

The soluble murine type I interferon receptor Ifnar-2 is present in serum, is independently regulated, and has both agonistic and antagonistic properties.

The ability to modify responses to type I interferons (IFNs) could alter processes such as hematopoiesis and immunity, which involve endogenous IFNs and responses to exogenous IFNs. The data presented here support a significant role for a recently identified soluble isoform of the murine type I IFN receptor, muIfnar-2a, as an efficient regulator of IFN responses. The messenger RNA (mRNA) transcript encoding muIfnar-2a is generally more abundant than that encoding the transmembrane isoform, muIfnar-2c. Furthermore, the ratio of muIfnar-2a:2c transcripts varied from more than 10:1 in the liver and other organs to less than 1:1 in bone-marrow macrophages, indicating independent regulation of the 2 transcripts encoding receptor isoforms and suggesting that the soluble muIfnar-2a levels are biologically relevant in some organs. Western blot analysis showed that soluble muIfnar-2 was present at high levels in murine serum and other biologic fluids and bound type I IFN. Recombinant muIfnar-2a competitively inhibited the activity of both IFNalpha and beta in reporter assays using the L929 cell line and in antiproliferative and antiviral assays using primary cells. Surprisingly, using primary thymocytes from Ifnar-2(-/-) mice, recombinant muIfnar-2a formed a complex with IFN alpha or beta and muIfnar-1 at the cell surface and transmitted an antiproliferative signal. These data indicate potential dual actions of soluble muIfnar-2 and imply that a signal can be transduced through the Ifnar-1 chain of the receptor complex in the absence of the cytoplasmic domain of Ifnar-2. Therefore, our results suggest that soluble Ifnar-2 is an important regulator of endogenous and systemically administered type I IFN.

SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine.

Mice lacking suppressor of cytokine signaling-1 (SOCS1) develop a complex fatal neonatal disease. In this study, SOCS1-/- mice were shown to exhibit excessive responses typical of those induced by interferon gamma (IFNgamma), were hyperresponsive to viral infection, and yielded macrophages with an enhanced IFNgamma-dependent capacity to kill L. major parasites. The complex disease in SOCS1-/- mice was prevented by administration of anti-IFNgamma antibodies and did not occur in SOCS1-/- mice also lacking the IFNgamma gene. Although IFNgamma is essential for resistance to a variety of infections, the potential toxic action of IFNgamma, particularly in neonatal mice, appears to require regulation. Our data indicate that SOCS1 is a key modulator of IFNgamma action, allowing the protective effects of this cytokine to occur without the risk of associated pathological responses.

Identification and characterization of two distinct truncated forms of gp130 and a soluble form of leukemia inhibitory factor receptor alpha-chain in normal human urine and plasma.

Leukemia inhibitory factor (LIF) is a polyfunctional cytokine known to require at least two distinct receptor components (LIF receptor alpha-chain and gp130) in order to form a high affinity, functional receptor complex. In this report, we present evidence that there are two distinct truncated forms of gp130 in normal human urine and plasma: a large form with a molecular weight of approximately 100, 000, which is similar to a previously described form of soluble gp130 in human serum, and a previously undescribed small form with a molecular weight of approximately 50,000. Using a panel of monoclonal antibodies raised against the extracellular domain of human gp130, we were able to show that the small form of the urinary gp130 probably contained only the hemopoietin domain. Both forms of gp130 bound LIF specifically and were capable of forming heterotrimeric complexes with soluble human LIF receptor alpha-chain in the presence of human LIF. In addition to the soluble forms of gp130, a soluble form of LIF receptor alpha-chain was also detected in human urine and plasma.

Cloning and characterization of soluble and transmembrane isoforms of a novel component of the murine type I interferon receptor, IFNAR 2.

This report describes the cloning of cDNAs encoding transmembrane and soluble isoforms of a novel chain of the murine type I interferon (IFN) receptor and characterization of its capability to bind ligand and transduce signals. The transmembrane receptor (murine IFNAR 2c) has an extracellular domain of 215 amino acids and an intracellular domain of 250 amino acids, with 48% amino acid and 71% nucleotide identity with human IFNAR 2c. The cDNA for the soluble murine receptor (IFNAR 2a) encodes a 221-amino acid polypeptide identical to the first 210 amino acids of IFNAR 2c plus a novel 11 amino acids. Northern blot analyses show that murine IFNAR 2 is expressed as two transcripts of 4 kilobases encoding the transmembrane isoform and 1.5 kilobases encoding the more abundant soluble isoform. Studies using primary murine cells that lack IFNAR 1 show that IFNAR 2 is expressed, and cells bind type I IFN ligand, but do not transduce signals as detected by electrophoretic mobility shift assays of ISGF3 or GAF complexes binding to their cognate oligonucleotides. These cells show no effects on the ability of IFNgamma to activate these complexes. These studies demonstrate that the IFNAR 2 transmembrane (2c) and soluble (2a) isoforms are conserved between the human and mouse and that IFNAR 2c has intrinsic ligand binding activity, but no intrinsic signal transducing activity as measured in this study.

The unusual species cross-reactivity of the leukemia inhibitory factor receptor alpha-chain is determined primarily by the immunoglobulin-like domain.

Human leukemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIFRs), while mouse LIF (mLIF) binds only to mouse LIFRs. Furthermore, hLIF binds with much higher affinity to the mouse LIFR (mLIFR) alpha-chain than does mLIF itself. To define the structural elements of the mLIFR alpha-chain conferring high affinity binding of hLIF and the species-specific interaction with mLIF, we first constructed C-terminally truncated extracellular domains of both the mLIFR and the human LIFR (hLIFR) alpha-chains, which contained only the two hemopoietin domains separated by an immunoglobulin-like domain. These recombinant truncated LIFR alpha-chains had identical binding and biological characteristics to either their naturally occurring or transfected counterparts. On the basis of this, we have generated eight interspecies receptor chimeras by combining different regions of the mouse and human LIFR sequence. Surprisingly, the immunoglobulin-like domain of the mLIFR alpha-chain played the predominant role in receptor-ligand interactions. Moreover, both high affinity binding for hLIF and the species-specific binding for mLIF mapped to the same domain of mLIFR molecule. These findings should enable the development of a "humanized" mouse LIFR that could act as a potent antagonist of hLIF biological activities in vivo.

Evidence for the formation of a heterotrimeric complex of leukaemia inhibitory factor with its receptor subunits in solution.

Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine that is known to require at least two distinct receptor components (LIF receptor alpha-chain and gp130) in order to form a high-affinity, functional, receptor complex. Human LIF binds with unusually high affinity to a naturally occurring mouse soluble LIF receptor alpha-chain, and this property was used to purify a stable complex of human LIF and mouse LIF receptor alpha-chain from pregnant-mouse serum. Recombinant soluble human gp130 was expressed, with a FLAG(R) epitope (DYKDDDDK) at the N-terminus, in the methylotropic yeast Pichia pastoris and purified using affinity chromatography. The formation of a trimeric complex in solution was established by native gel electrophoresis, gel-filtration chromatography, sedimentation equilibrium analysis, surface plasmon resonance spectroscopy and chemical cross-linking. The stoichiometry of this solution complex was 1:1:1, in contrast with that of the complex of interleukin-6, the interleukin-6-specific low-affinity receptor subunit and gp130, which is 2:2:2.

A type I interferon signaling factor, ISF21, encoded on chromosome 21 is distinct from receptor components and their down-regulation and Is necessary for transcriptional activation of interferon-regulated genes.

The type I interferons (IFNs) are a family of cytokines, comprising at least 17 subtypes, which exert pleiotropic actions by interaction with a multi-component cell surface receptor and at least one well characterized signal transduction pathway involving JAK/STAT (Janus kinase/signal transducer and activator of transcription) proteins. In a previous report, we showed that a signaling factor, encoded by a gene located on the distal portion of chromosome 21, distinct from the IFNAR-1 receptor, was necessary for 2'-5'-oligoadenylate synthetase activity and antiviral responses, but not for high affinity ligand binding. In the present studies using hybrid Chinese hamster ovary cell lines containing portions of human chromosome 21, we show that the type I IFN signaling molecule, designated herein as ISF21, is distinct from the second receptor component, IFNAR-2, which is expressed in signaling and non-signaling cell lines. The location of the gene encoding ISF21 is narrowed to a region between the 10;21 and the r21 breakpoints, importantly eliminating the Mx gene located at 21q22.3 (the product of which is involved in IFN-induced antiviral responses) as a candidate for the signaling factor. To characterize the action of this factor in the type I IFN signaling pathway, we show that it acts independently of receptor down-regulation following ligand binding, both of which occur equally in the presence or absence of the factor. In addition, we demonstrate that ISF21 is necessary for transcriptional activation of 2'-5'-oligoadenylate synthetase, 6-16, and guanylate-binding protein gene promoter reporter constructs, which are mediated by several signaling pathways. ISF21 represents a novel factor as the localization to chromosome 21, and the data presented in this study exclude any of the known type I IFN signal-transducing molecules.

Molecular basis of the soluble and membrane-bound forms of the murine leukemia inhibitory factor receptor alpha-chain. Expression in normal, gestating, and leukemia inhibitory factor nullizygous mice.

The murine leukemia inhibitory factor receptor alpha-chain (mLIFR) exists in a membrane-bound and a soluble form. The two major classes of mRNA transcript correspond to either the soluble or membrane-bound form of the mLIFR. In this study we have identified a complex and heterogeneous pattern of expression of mRNA transcripts for this receptor in normal mouse tissues and cell lines. In order to understand the molecular basis of these transcripts, genomic clones encompassing the region of divergence from the soluble to the membrane-bound form of the receptor were isolated. cDNAs encoding the membrane-bound form of the mLIFR were generated by an alternative splicing event where an exon that is specific to the soluble mLIFR was skipped. The membrane-bound form of the mLIFR was heterogeneously polyadenylated with at least five different sites of polyadenylation. The mRNA transcript encoding the soluble form of the mLIFR contained a region highly homologous to a murine B2 repetitive element, thus providing a possible explanation for the genesis of this transcript. The different forms of the mLIFR were analyzed in a wide range of mouse tissues in pseudopregnant mice and in mice at various stages of pregnancy. Only liver, placenta, and uterus showed an increase in the levels of mLIFR mRNA expression during pregnancy, indicating an important role for the LIFR in this process. However, somewhat surprisingly, there was no detectable difference in mLIFR mRNA levels or levels of soluble protein in leukemia inhibitory factor nullizygous mice when compared with normal mice.

Conversion of the biological specificity of murine to human leukemia inhibitory factor by replacing 6 amino acid residues.

Mouse and human leukemia inhibitory factor (mLIF and hLIF) have approximately 80% amino acid identity, but mLIF cannot bind to the hLIF receptor (hLIF-R), while hLIF binds to the alpha-chain of the mLIF receptor (mLIF-R) with a much higher affinity than does mLIF. We have previously shown that the same regions confer both these properties of hLIF and map to an area within the predicted third alpha-helix and two of the loops of hLIF (Owczarek, C. M., Layton, M. J., Metcalf, D., Lock, P., Wilson, T. A., Gough, N. M., and Nicola, N. A. (1993) EMBO J. 12, 3487-3495). The present studies, using interspecies chimeras of mLIF and hLIF, have defined 6 residues (Asp57, Ser107, His112, Ser113, Val155, and Lys158) that contribute most of the binding energy involved in the interaction of hLIF and the hLIF-R alpha-chain, and form a surface at one end of the predicted four alpha-helical bundle of the hLIF molecule. Mouse LIF is unable to bind to the hLIF-R alpha-chain or activate the cellular hLIF-R, but when these 6 residues were substituted onto an mLIF framework, they were able to reconstitute both the binding and biological activities specific to hLIF.

Homology modelling and 1H NMR studies of human leukaemia inhibitory factor.

Human leukaemia inhibitory factor (LIF) is a glycoprotein with a diverse range of activities on many cell types. A molecular model of LIF has been constructed based mainly on the structure of the related cytokine granulocyte colony-stimulating factor, and refined using simulated annealing and molecular dynamics in water. The model was stable during molecular dynamics refinement and is consistent with known stereochemical data on proteins. It has been assessed by comparison with 1H NMR data on the ionization behaviour of the six histidine residues in LIF, the imidazolium pKa values of which range from 3.6 to 7.4. These pKa values were assigned to individual histidine residues from NMR studies on a series of His-->Ala mutants. The environments of the histidine residues in the model account very well for their observed ionization behaviour. Furthermore, the model is consistent with mutagenesis studies which have defined a group of amino acid residues involved in receptor binding.

Complex binding of leukemia inhibitory factor to its membrane-expressed and soluble receptors.

The complex interaction of leukemia inhibitory factor (LIF) with its specific receptor present on the cell surface, in isolated membranes and in solution, has been examined in detail. Several aspects of this complexity have been highlighted, including the presence of high- and low-affinity murine LIF receptors, biphasic dissociation of human LIF from apparently homogeneous high- or low-affinity human LIF receptors, and unusual species cross-reactivity. The unusual species cross-reactivity observed between murine and human LIF has also been exploited to map an important receptor binding epitope on human LIF.

NMR studies of a murine-human chimera of leukaemia inhibitory factor (LIF). Comparison with human LIF.

Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine active on many cell types. We present here 1H NMR studies on the solution properties and stability of MH35, a chimera of murine and human LIF which can be expressed at high levels in Escherichia coli, thus enabling efficient labelling of the protein with the stable isotopes 13C and 15N. MH35 has 85% sequence identity with human LIF and similar activity in biological assays. The 1H chemical shifts of the 12 conserved aromatic residues and the pKa values of the five conserved histidine residues in MH35 and human LIF are very similar. Temperature dependence studies indicate that both proteins are stable, with significant conformational changes occurring only above 70 degrees C. These results show that these proteins have a similar overall structure and stability and that MH35 is therefore a suitable analogue of human LIF for structural studies in solution.