Functional inhibition of PI3K by the betaGBP molecule suppresses Ras-MAPK signalling to block cell proliferation.

The mechanisms of signal transduction from cell surface receptors to the interior of the cell are fundamental to the understanding of the role that positive and negative growth factors play in cell physiology and in human diseases. Here, we show that a functional link between phosphatidylinositol-3-OH kinase (PI3K) and Ras is suppressed by the beta-galactoside binding protein (betaGBP) molecule, a cytokine and a negative cell-cycle regulator. Ras-mitogen-activated protein kinase (MAPK) signalling is blocked by betaGBP owing to its ability to inhibit the p110 catalytic subunit of PI3K, whose basal activity is required for Ras activation. Functional inhibition of p110 by betaGBP results in downregulation of PI3K activity, suppression of Ras-GTP loading, consequent loss of MAPK activation and block of cell proliferation. This study sheds light on the molecular mechanisms whereby betaGBP can control cell proliferation and, by extension, may potentially control tumorigenesis by controlling PI3K.

Cell cycle arrest and induction of apoptosis by beta galactoside binding protein (beta GBP) in human mammary cancer cells. A potential new approach to cancer control.

Conflict between mitogenic pressure, as is the case in tumour cells and an imposed inability to proceed through the cell cycle may result in cell death. In the present study we examined the effect of beta galactoside binding protein (beta GBP), a negative growth factor which controls cell cycle transition from S phase into G2, on three human mammary cell lines which differ for oncogenic potential, oestrogen receptor expression and expression of the EGF receptor family. We found that in all cases beta GBP induced a cell cycle block prior to the cells' entry into G2 and that this was followed by progressive apoptotic death. This evidence on epithelial cancer cells parallels previous data on tumour cells of mesenchymal origin and suggests that beta GBP has potential therapeutic implications in the treatment of cancers.

Negative cell cycle control of human T cells by beta-galactoside binding protein (beta GBP): induction of programmed cell death in leukaemic cells.

The cell cycle is negatively regulated by diverse molecular events which originate in part from the interaction of secreted proteins with specific cell surface receptors. By exerting negative control on cell proliferation, these factors can help maintain cell number balance both through growth restraints and the induction of apoptosis and may thus contribute to prevent or control tumourigenesis. Here we report that betaGBP, a negative growth factor which controls transition from S phase into G2, causes an S/G2 growth arrest in both normal and leukaemic T cells. However, in leukaemic T cells but not in normal T lymphocytes, growth arrest is followed by apoptosis. Analysis of possible mechanisms of induction of apoptosis does not support Fas and Fas L as having a main role but points instead to Bcl-2 and Bax. The induction of apoptosis in leukaemic T cells is characterised by the decrease of Bcl-2 and consequent predominance of Bax. By contrast, in the normal T cells, which do not enter apoptosis, the quantitative relationship of Bcl-2 to Bax remains unchanged. The ability of betaGBP to selectively induce apoptosis in leukaemic cells suggests that betaGBP may play a role in cancer surveillance and that its use has potential therapeutic implications.

Beta-galactoside-binding protein (beta GBP) alters the cell cycle, up-regulates expression of the alpha- and beta-chains of the IFN-gamma receptor, and triggers IFN-gamma-mediated apoptosis of activated human T lymphocytes.

In this paper, the effects of beta-galactoside binding protein (beta GBP), the LGALS1 gene product, on the cell cycle progression and expansion of activated human T lymphocytes were studied. Beta GBP drastically inhibits the IL-2 induced proliferation of PHA-activated T lymphocytes as well as the IL-2 independent proliferation of malignant T lymphocytes by arresting them in the S and G2/M phases of the cell cycle. In addition, beta GBP up-regulates the expression of both the alpha- and the beta-chains of the IFN-gamma R on activated T lymphocyte membrane. None of these effects depend on sugar binding: saturating amounts of lactose do not affect the cell cycle block nor IFN-gamma R up-modulation. The increased expression of both chains renders beta GBP-treated T lymphoblasts sensitive to IFN-y-induced apoptosis. Taken as a whole, these findings suggest that beta GBP plays an important immunoregulatory role by switching off T lymphocyte effector functions. They also provide the first evidence of up-modulation of IFN-gamma R expression on T lymphocytes by a negative cell growth regulator.

Beta-galactoside-binding protein secreted by activated T cells inhibits antigen-induced proliferation of T cells.

We have used mRNA differential display PCR to search for genes induced in activated T cells and have found the LGALS1 (lectin, galactoside-binding, soluble) gene to be strongly up-regulated in effector T cells. The protein coded by the LGALS1 gene is a beta-galactoside-binding protein (betaGBP), which is released by cells as a monomeric negative growth factor but which can also associate into homodimers (galectin-1) with lectin properties. Northern blot analysis revealed that ex vivo isolated CD8+ effector T cells induced by a viral infection expressed high amounts of LGALS1 mRNA, whereas LGALS1 expression was almost absent in resting CD8+ T cells. LGALS1 expression could be induced in CD4+ and CD8+ T cells upon activation with the cognate peptide antigen and high levels of LGALS1 expression were found in concanavalin A-activated T cells but not in lipopolysaccharide-activated B cells. Gel filtration and Western blot analysis revealed that only monomeric betaGBP was released by activated CD8+ T cells and in vitro experiments further showed that recombinant betaGBP was able to inhibit antigen-induced proliferation of naive and antigen-experienced CD8+ T cells. Thus, these data indicate a role of betaGBP as an autocrine negative growth factor for CD8+ T cells.

Characterisation and antiproliferative activity of an alpha-type murine interferon from embryonic fibroblasts.

Interferons play a part in the negative control of cell proliferation of mammalian cells. Here a natural interferon has been isolated from soluble proteins secreted by secondary murine embryonic fibroblasts using Blue Sepharose chromatography, immunoaffinity exclusion and Q Sepharose ion exchange fractionation. Partial amino acid sequencing assigns it to the interferon alpha family. Its biological and physico-chemical properties single it out from all other murine alpha interferons. The embryonic interferon has stronger antiproliferative activity, is acid labile, has stronger affinity for Blue Sepharose and weak affinity for antibodies which recognise other murine interferon alpha subtypes.

Molecular expression of the negative growth factor murine beta-galactoside binding protein (mGBP).

Characterisation of the negative growth factor mGBP at molecular and biological levels indicates that the protein has no lectin nature and suggests instead a participation in the cytokine network. The protein is shown to be expressed as a monomer in two forms, one of which is non-covalently linked to a glycan complex. This confers greater efficiency to the inhibitor and may favour a paracrine role. The two monomeric forms may oxidise into tetramers which retain biological activity, but lack ability to link to specific saccharide residues.

Cell cycle regulation (G1) by autocrine interferon and dissociation between autocrine interferon and 2',5'-oligoadenylate synthetase expression.

In tertiary MEF undergoing cell cycle progression, autocrine interferon (IFN) is released and constitutive levels of 2',5'-oligoadenylate (2-5A) synthetase activity, low through the cell cycle, surge into a peak within S phase. Treatment of MEF with the autocrine IFN they produce elicits a 2-5A synthetase response from cells positioned in G0 but not from cells in G1 and from cells in S phase. Neutralization of the autocrine IFN by antibody shortens the length of G1 leaving unaltered the kinetics of progression through S and G2 and has no effect on the S phase-linked expression of 2-5A synthetase activity. The growth controlling effect of the autocrine IFN has been mapped to the second part of G1.

Structure and expression of the negative growth factor mouse beta-galactoside binding protein gene.

Following the identification of murine beta-galactoside binding protein (mGBP) as an autocrine negative growth factor we have now isolated and characterized the genomic region spanning the mGBP gene and have determined the 5' end of the transcript by primer extension, S1 mapping and mRNA sequence. The gene is found to be contained within 4 kilobases and composed of four exons of 79, 80, 171 and 197 nucleotides separated by three introns of 1200, 1600 and 193 nucleotides. The DNA region upstream of the 5' end of the transcript contains canonical sequences for eukaryotic promoter elements including CAT and TATA boxes and several DNA motifs for potential transcription regulation. The gene is differentially expressed in a variety of normal tissues.

Identification of an autocrine negative growth factor: mouse beta-galactoside-binding protein is a cytostatic factor and cell growth regulator.

Murine beta-galactoside-binding protein, a protein classified as a soluble lectin, is shown to be a cell growth-regulatory molecule and a cytostatic factor. The growth-inhibitory effect is not related to lectin properties, and competition assays indicate that the protein binds to specific cell surface receptors with high affinity. It exerts control in G0 and at G2, both as a regulator of cell replication and as a cytostatic factor.

Cell cycle regulation (G1) by autocrine interferon and dissociation between autocrine interferon and 2',5'-oligoadenylate synthetase expression.

In tertiary MEF undergoing cell cycle progression, autocrine interferon (IFN) is released and constitutive levels of 2',5'-oligoadenylate (2-5A) synthetase activity, low through the cell cycle, surge into a peak within S phase. Treatment of MEF with the autocrine IFN they produce elicits a 2-5A synthetase response from cells positioned in G0 but not from cells in G1 and from cells in S phase. Neutralization of the autocrine IFN by antibody shortens the length of G1 leaving unaltered the kinetics of progression through S and G2 and has no effect on the S phase-linked expression of 2-5A synthetase activity. The growth controlling effect of the autocrine IFN has been mapped to the second part of G1.

Cell cycle position and expression of encephalomyocarditis virus in mouse embryo fibroblasts.

Infection of mouse embryo fibroblasts in G1 or S phase with encephalomyocarditis virus gave different kinetics of viral RNA synthesis. In S phase cells, RNA synthesis was faster and reached higher levels than in G1 cells. Virus-specified proteins were fewer in G1 cells than in S cells during the early stage of the infection and c.p.e. in G1 cells appeared about 4 h later than in S cells. Addition of a cellular factor with ability to affect cell conformation had an inhibitory effect on viral RNA synthesis.

Expression of the 2-5A system during the cell cycle.

To determine whether the 2-5A system has a role in the regulation of cell growth we have examined all constituents of the 2-5A pathway in mouse embryo fibroblasts undergoing one cycle of division at the tertiary stage under conditions where a high degree of uniformity is maintained within each stage of the cycle. Levels of the 2-5A synthetase increased up to tenfold late in S phase and declined as cells moved through G2. A similar but smaller increase in the 2-5A-dependent ribonuclease was observed, whereas activity of the 2'5' phosphodiesterase was highest in quiescent cells. At the time of maximum synthetase levels no phosphorylated 2-5A could be detected in the intact cell. Endogenous interferon (IFN) was found in the culture supernatants in increasing concentration with cell cycle progression and addition of antibodies to IFN reduced the increase in synthetase seen in late S. Treatment of cells with a growth inhibitor that cells produce also affected synthetase activity.

Cell surface and cytokinetic structures in cell shape determination of mouse fibroblasts. Events involved in cell rounding induced by removal of loosely bound macromolecular moieties from the cell surface.

Cell rounding was induced in synchronised mouse embryo fibroblasts without the use of drugs by a new method based on removal from the cell surface of loosely bound macromolecular moieties. The cells were examined by scanning and transmission electron microscopy and also by specific fluorescence methods during experimentally induced progression from the spread to a rounded form to assess changes in the cell surface and in the arrangement of cytoskeletal elements. Early events were a loss of order in the subcortical microfilament network and the formation of surface blebs. This was followed by disintegration of stress fibres and body retraction. Microtubules and intermediate filaments maintained their integrity but moved inwards towards the rounded perinuclear part of the cells. The relationship between surface alterations, organisation of the cytoskeleton and cell conformation is discussed.

Properties of a cell growth inhibitor produced by mouse embryo fibroblasts.

Secondary mouse embryo fibroblasts produce a growth inhibitor with the character of a thermolabile, nondialysable protein. The inhibitor was harvested from conditioned medium, and following G-75 Sephadex fractionation it was isolated in one peak which consisted of two fractions eluting at approximately two thirds of the bed volume of the column where approximately 80 percent of the original activity was recovered with an increase in specific activity of about tenfold. Polyacrylamide gradient gel electrophoresis of fractions from L-[35S] methionine-labelled conditioned medium showed that the two fractions with growth inhibitory activity contained some 4-5 bands and shared the two major components. Cell cycle studies showed that the growth inhibitory effect was exerted after addition during early and late G1 and during S phase, and morphological studies showed that where growth was inhibited the morphological expression of the cells was altered.

Cell cycle study on the effect of interferon on synchronized mouse embryo fibroblasts.

Mouse embryo fibroblasts synchronized by controlling cultural conditions were used to examine the effects of interferon (IFN) while undergoing a single synchronous cycle of division at the tertiary stage. IFN was added early in G1 and at the G1-S boundary and the duration of specific phases of the cycle were investigated together with biochemical events related to cell cycle progression. Assessment of population distribution by fluorimetric quantitation of DNA content showed that IFN extended G1 and G2 but had no effect on the duration of S phase. Assessment of transport and uptake of exogenous TdR and measurements of specific kinase activity under conditions where DNA synthesis and S phase were not altered showed that IFN had no effect on TdR transport but could markedly reduce TdR uptake, and delay the S phase associated increase of TdR-kinase activity.

Influence of the cytoskeleton on the expression of a mouse hepatitis virus (MHV-3) in peritoneal macrophages: acute and persistent infection.

The effects of anti-cytokinetic drugs on virus production, formation of syncytia, cell surface changes and lysosomal damage were examined during mouse hepatitis virus 3 (MHV-3) infection of mouse peritoneal macrophages. Colchicine and vinblastine caused no detectable effect on the infectious process. In the presence of cytochalasin B the acute, highly cytopathogenic interaction that MHV-3 establishes with macrophages was converted into one which persisted for several days. Under these conditions the cell surface changes induced by the infection were maintained unaltered but cell fusion was reduced and no significant lysosomal damage was detectable.

A cell cycle study of the effects of Con A on synchronized mouse embryo fibroblasts: arrest at two specific stages of the cycle and dissociation between uptake of thymidine and DNA synthesis.

We have examined the effects of 50 microgram ml-1 of Con A added to synchronized mouse embryo fibroblasts at different times during the cell cycle. We found that Con A caused arrest of growth not solely by preventing G1-G0 cells from entering the S-phase but also by exerting a G2 block. We also found that Con A, which prevented commencement of S-phase, did not arrest cells already in S from reaching the G2 stage but inhibited the S-phase associated process of thymidine uptake. The inhibition was greater when the Con A receptors were extensively clustered.