Valerie Wells

β-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 β‐galactoside‐binding protein (βGBP), 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 βGBP was released by activated CD8+ T cells and in vitro experiments further showed that recombinant βGBP was able to inhibit antigen‐induced proliferation of naive and antigen‐experienced CD8+ T cells. Thus, these data indicate a role of βGBP as an autocrine negative growth factor for CD8+ T cells.

Identification of an autocrine negative growth factor: Mouse β-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 arrest and induction of apoptosis by β galactoside binding protein (βGBP) 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 β galactoside binding protein (βGBP), 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 βGBP 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 βGBP 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 βGBP, 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 βGBP to selectively induce apoptosis in leukaemic cells suggests that βGBP may play a role in cancer surveillance and that its use has potential therapeutic implications. J Cell Physiol 178:102–108, 1999.

Molecular expression of the negative growth factor murine β-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.

Circumventing Multidrug Resistance in Cancer by β-Galactoside Binding Protein, an Antiproliferative Cytokine

We report here that beta-galactoside binding protein (betaGBP), an antiproliferative cytokine which can program cancer cells to undergo apoptosis, exhibits equal therapeutic efficacy against cancer cells that display diverse mechanisms of drug resistance and against their parental cells. The mechanisms of drug resistance in the cancer cells that we have examined include overexpression of P-glycoprotein, increased efficiency of DNA repair, and altered expression and mutation in the topoisomerase I and II enzymes. We also report that betaGBP exerted its effect by arresting the cells in S phase prior to the activation of programmed cell death. The uniquely similar profile of response to betaGBP by these drug-resistant cells and their parental cells extends the therapeutic potential of this cytokine in the treatment of cancers and offers a promising alternative to patients whose tumors are refractory to the currently available cadre of chemotherapeutic agents.

Phosphoinositide 3-kinase targeting by the β galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death

IntroductionPhosphoinositide 3-kinase (PI3K)-activated signalling has a critical role in the evolution of aggressive tumourigenesis and is therefore a prime target for anticancer therapy. Previously we have shown that the β galactoside binding protein (βGBP) cytokine, an antiproliferative molecule, induces functional inhibition of class 1A and class 1B PI3K. Here, we have investigated whether, by targeting PI3K, βGBP has therapeutic efficacy in aggressive breast cancer cells where strong mitogenic input is fuelled by overexpression of the ErbB2 (also known as HER/neu, for human epidermal growth factor receptor 2) oncoprotein receptor and have used immortalised ductal cells and non-aggressive mammary cancer cells, which express ErbB2 at low levels, as controls.MethodsAggressive BT474 and SKBR3 cancer cells where ErbB2 is overexpressed, MCF10A immortalised ductal cells and non-invasive MCF-7 cancer cells which express low levels of ErbB2, both in their naive state and when forced to mimic aggressive behaviour, were used. Class IA PI3K was immunoprecipitated and the conversion of phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) assessed by ELISA. The consequences of PI3K inhibition by βGBP were analysed at proliferation level, by extracellular signal-regulated kinase (ERK) activation, by akt gene expression and by apoptosis. Apoptosis was documented by changes in mitochondrial membrane potential, alteration of the plasma membrane, caspase 3 activation and DNA fragmentation. Phosphorylated and total ERK were measured by Western blot analysis and akt mRNA levels by Northern blot analysis. The results obtained with the BT474 and SKBR3 cells were validated in the MCF10A ductal cells and in non-invasive MCF-7 breast cancer cells forced into mimicking the in vitro behaviour of the BT474 and SKBR3 cells.ResultsIn aggressive breast cancer cells, where mitogenic signalling is enforced by the ErbB2 oncoprotein receptor, functional inhibition of the catalytic activity of PI3K by the βGBP cytokine and loss of akt mRNA results in apoptotic death. A functional correlation between ERK and the kt gene was also found. The relationship between ERK, akt mRNA, PI3K and cell vulnerability to βGBP challenge was sustained both in mammary ductal cells forced to mimic an aggressive behaviour and in non-aggressive breast cancer cells undergoing an enforced shift into an aggressive phenotype.ConclusionsβGBP, a newly discovered physiological inhibitor of PI3K, is a selective and potent inducer of apoptosis in aggressive breast cancer cells. Due to its physiological nature, which carries no chemotherapeutic disadvantages, βGBP has the potential to be safely tested in clinical trials.

Tregs utilize β-galactoside-binding protein to transiently inhibit PI3K/p21ras activity of human CD8+ T cells to block their TCR-mediated ERK activity and proliferation

Regulatory T cells (Tregs) and beta-galactoside-binding protein (betaGBP), a regulatory protein often found expressed at sites of immunological privilege, have similar functions. Their presence affects the outcome of harmful autoimmunity and cancers, including experimental autoimmune encephalomyelitis and malignant gliomas. Here we report a novel pathway by which Tregs express and utilize betaGBP to control CD8(+) T cell responses partially activating TCR signaling but blocking PI3K activity. As a result, this leads to a loss of p21(ras), ERK and Akt activities despite activation of TCR proximal signals, such as phosphorylation of CD3zeta, Zap70, Lat and PKCtheta. Although non-processive TCR signaling often leads to cell anergy, Tregs/betaGBP did not affect cell viability. Instead, betaGBP/Tregs transiently prevented activation of CD8(+) T cells with self-antigens, while keeping their responses to xenogeneic antigens unaffected.

Turning cell cycle controller genes into cancer drugs: A role for an antiproliferative cytokine (βGBP)

Cancer therapies based on drugs designed to interfere with specific targets within the molecular circuitry of cancer cells are currently under intense experimentation. Our strategy is based on the use of a naturally occurring immunomolecule which can selectively kill cancer cells, based on its ability to exploit genetic differences between normal and cancer cells. The betaGBP cytokine has previously been shown to negatively regulate the cell cycle by blocking cells in late S phase. In tumour cells, but not in normal cells, the S phase block has been shown to be followed by apoptosis. Mechanisms involved in S phase arrest have been pinpointed to downregulation of signalling and altered expression of cell cycle controller proteins, including E2F1, a transcription factor with ability to play a part in apoptosis. Here we discuss the use of betaGBP within the context of cancer surveillance and cancer therapeutics focussing on E2F1 as one mechanistic aspect relevant to betaGBPs selective induction of programmed cell death in cancer.

Negative Control of Cell Proliferation. Growth Arrest versus Apoptosis. Role of βGBP

βGBP is a novel physiological negative growth regulator of the cell and a cytostatic factor.It is secreted by cells, and bybinding with high affinity to specific cell surface receptors. In normal cell surface receptors. In normal cells, βGBP physiologically controls transition from G0 to G1 and passage from late S phase to G2 by modulating signalling cascades activated by tyrosine Kinase receptors and by affecting transcription events.As a cytostatic Factor βGBP has a marked growth inhibitory effect on a variety of tumours including leukaemias where growth arrest is followed by the activation of apoptotic pathways and cell death.