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

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Chick embryo cells: These were obtained from 10-day-old chicks and grown in a tris0ay-droxymethyl)aminomethane-bffffered Gey's solution with peptone and lactalbumin hydrolysate (4). They were infected after 2 days' growth on coverslips or in 6 mm diameter Petri dishes.HeLa cdls: These were propagated in medium 199 plus 5 per cent inactivated calf serum and were infected 3 to 6 days after subculture by trypsinization. 463

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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 b-galactoside binding protein (bGBP) molecule, a cytokine and a negative cell-cycle regulator. Ras-mitogen-activated protein kinase (MAPK) signalling is blocked by bGBP 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 bGBP 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 bGBP can control cell proliferation and, by extension, may potentially control tumorigenesis by controlling PI3K. The Ras-mitogen-activated protein kinase (MAPK) signalling pathway plays a key role in the activation dynamics of transcriptional events required for mammalian cells to enter the cell cycle. Activation of Ras requires growth factor/receptor tyrosine kinase (RTK) interaction, RTK cross-phosphorylation leading to the formation of high-affinity binding sites for SH2 domains of the Grb2 and Shc adapter proteins, the binding of the nucleotide exchange factor SOS to the SH3 domains of Grb2 and the conversion by SOS of GDP-Ras into active GTP-Ras. The ensuing signalling cascade leads to the phosphorylation of MAPK and the activation of genes programmed to initiate cell proliferation (Downward, 2003;Mitin et al., 2005 to B5 Â 10 4 receptors/cell to control cell-cycle entry and S/G 2 traverse in normal cells Mallucci, 1991, 1992). As a cytostatic factor, bGBP induces reversible S/G 2 arrest in normal cells while activating programmed cell death in cancer cells Mallucci et al, 2003). We now report that in murine embryonic fibroblasts (MEF), chosen as a paradigm of normal cells, bGBP inhibits phosphatidylinositol-3-OH kinase (PI3K) activity, also that PI3K activity is necessary for growth factor-induced activation of Ras and that basal levels are sufficient. Comparative studies with PI3K inhibitors indicate that the p110 catalytic subunit of PI3K is a prime target of bGBP-induced signalling.To determine the mode by which bGBP inhibits cell proliferation, we have investigated mechanistic events within the signalling context that lead to the phosphorylation and activation of transcription factors involved in cell-cycle entry and cell proliferation. We found (data not shown) that in MEF from secondary culture, held in G 0 by 20 nM Hu-r-bGBP and stimulated with either 10 ng/ml platelet derived growth factor (PDGF), 10 ng/ ml epidermal growth factor (EGF), 5 ng/ml fibroblast growth factor (FGF) or 10% fetal calf serum, there was no evidence of c-fos, c-jun nor, predictably, of c-myc mRNA expression indicating that no activating signals had reached the pr...