Effect Of Gal-1 Research Articles

Galectin-1: biphasic growth regulation of Leydig tumor cells

Galectin-1 (Gal-1) is a widely expressed beta-galactoside-binding protein that exerts pleiotropic biological functions. To gain insight into the potential role of Gal-1 as a novel modulator of Leydig cells, we investigated its effect on the growth and death of MA-10 tumor Leydig cells. In this study, we identified cytoplasmic Gal-1 expression in these tumor cells by cytofluorometry. DNA fragmentation, caspase-3, -8, and -9 activation, loss of mitochondrial membrane potential (DeltaPsim), cytochrome c (Cyt c) release, and FasL expression suggested that relatively high concentrations of exogenously added recombinant Gal-1 (rGal-1) induced apoptosis by the mitochondrial and death receptor pathways. These pathways were independently activated, as the presence of the inhibitor of caspase-8 or -9 only partially prevented Gal-1-effect. On the contrary, low concentrations of Gal-1 significantly promoted cell proliferation, without inducing cell death. Importantly, the presence of the disaccharide lactose prevented Gal-1 effects, suggesting the involvement of the carbohydrate recognition domain (CRD). This study provides strong evidence that Gal-1 is a novel biphasic regulator of Leydig tumor cell number, suggesting a novel role for Gal-1 in the reproductive physiopathology.

Galectin-1 Influences Migration of Retinal Pigment Epithelial Cells

To determine whether the beta-galactoside-binding matricellular protein Gal-1 is expressed in human specimens of proliferative vitreoretinopathy (PVR) and to evaluate its influence on RPE migration. RT-PCR was used to detect Gal-1-specific transcripts in PVR membranes, and the expression pattern of Gal-1 was examined by immunohistochemistry. Expression of Gal-1 in native, low- and high-density cultured RPE cells was determined by Western blot analysis. Cultured human RPE cells were treated with bFGF, TGF-beta2, PDGF-BB, or HGF. The dose-response of Gal-1 mRNA expression was measured by by real-time quantitative RT-PCR and Northern blot analysis. Induction of Gal-1 protein was confirmed by Western blot analysis. To study the effect of Gal-1 on RPE migration in vitro, Gal-1 expression was silenced by RNA interference. beta-Lactose was used to saturate extracellular galectins. RPE cell migration was assessed by a modified Boyden chamber assay, with HGF as the chemoattractant. Gal-1 mRNA expression was present in human specimens of PVR membranes, and staining for Gal-1 was distributed throughout the extracellular matrix (ECM) of PVR membranes. Colocalization was found with laminin and fibronectin and cells of epithelial origin. Western blot analysis revealed greater baseline expression levels in low-density cultured RPE cells than in native and high-density cultured RPE cells. Treatment with HGF caused a dose-dependent increase in Gal-1 expression. Low expression levels of Gal-1 correlated with a reduction of RPE migration to 14% of control. beta-Lactose inhibited HGF-induced RPE cell migration to 23% of control. Gal-1 is present in the extracellular matrix of PVR membranes and may be derived from dedifferentiated RPE cells. The expression level of Gal-1 appears to be related to a migratory RPE phenotype and stimulation by HGF, both conditions implicated in the pathogenesis of early PVR. Furthermore, HGF-induced RPE migration may be dependent, at least in part, on Gal-1- and beta-galactoside-dependent mechanisms.

Galectin-1 Can Support the Survival of Human Myeloma Cells in In Vitro Culture.

Myeloma cells as well as other hematopoietic cells exist in the bone marrow (BM), where adhesion molecules, cytokines and chemokines may play very important roles in regulating the survival, proliferation and lodgment of myeloma cells.Galectin-1(Gal-1) is a 14 KDa protein with the binding affinity to the molecules which carry β galactosides in their structure such as CD3, CD4, CD7, CD43, CD45 and integrins. Although Gal-1 is considered to be ubiquitously present in the BM, it remains to be clarified what is the effect of Gal-1 on the survival of myeloma cells. First, we confirmed that Gal-1 as well as SDF-1 was ubiquitously present in the extracellular space of BM samples from MM patients by the immunohistochemical staining.Furthermore, the expression of Gal-1 gene was found in a BM stromal cell line, KM-102, and also in primary myeloma cells as well as myeloma cell lines, while the expression of SDF-1 gene was detected in KM-102 cells but not in myeloma cells. In the serum-free culture in vitro, Gal-1 could support the survival of primary myeloma cells in the combination with IL-6 and SDF-1 at least for 2 weeks. In order to clarify the mechanism of the effect of Gal-1, we examined the effect of Gal-1 on the survival of myeloma cell lines and B cell l,ines; Ga-1 could support the survival of CD45- U266 and KMS-5 cells, but not support and suppress the survival of B cell lines (Raji and KUS cells) which express CD45, CD45RA and CD45RB but not CD45RO. By using the transfectants of CD45RA, CD45RB or CD45RO in U266 cells, we confirmed that Gal-1 could suppress the survival of CD45RA+ U266 cells as well as B cell lines but not of CD45RB or CD45RO transfectants of U266 cells. These data confirmed that Gal-1 could bind to the CD45RA molecule more effectively than CD45RB or CD45RO. We also revealed that Gal-1 stimulation inhibited the phosphorylation of ERK1/2 in CD45RA+ U266 cells as well as B cell lines, while in CD45- myeloma cells as well as CD45RO+ myeloma cells Gal-1 induced the activation of Akt. Therefore, these data suggest that Gal-1 plays an important role in the supporting the survival of myeloma cells but not B cells in the BM microenvironment.

Purification of human galectin-1 produced in high-cell density cultures of recombinant Escherichia coli: a comparison with classic shake flask cultivation

The aim of the present work was to develop a highly productive and simplified process for active human galectin-1 (Gal1) production. Gal1 is a β-galactoside binding lectin that differentially affects biological and cellular functions such as immune surveillance and apoptosis. These effects have attracted the attention of researchers in cell biology, biochemistry and immunology. However, the production of sufficient amounts of recombinant human Gal1 (rhGal1) is needed to study of the effects of Gal1 during cell treatments. To this end, an high-yield expression of rhGal1 was achieved by high-cell density fed-batch cultivation using an exponential glycerol feeding strategy and rhGal1 was purified by a one-step purification scheme using affinity chromatography.

Binding of galectin-1 (gal-1) on trophoblast cells and inhibition of hormone production of trophoblast tumor cells in vitro by gal-1.

Galectin-1 (gal-1), a member of the mammalian beta-galactoside-binding proteins, recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. As demonstrated histochemically, the lectin recognizes appropriate glycoepitopes on the syncytiotrophoblast and on chorionic carcinoma cells (BeWo). Freshly isolated trophoblast cells and trophoblast tumor cells Jeg3 did not bind gal-1. BeWo cells in contrast to Jeg3 form a syncytium in vitro and synthesize progesterone as well as hCG. BeWo cells were used as an approach to study the effects of gal-1 on hormone production. The lectin decreased cellular hCG and progesterone production as well as hCGbeta gene transcription as measured by real-time RT-PCR. Gal-1 mediated inhibition of cellular progesterone production was reduced in the presence of a Thomsen-Friedenreich (TF)-polyacrylamide conjugate. Inhibition of cellular hCG and progesterone production was also induced by anti-TF monoclonal antibodies. The results demonstrate that ligation of Galbeta1-4GlcNAc and Galbeta1-3GalNAc (TF) epitopes on BeWo cells may have regulatory effects on hCG and progesterone production.

Galectin-1, a cell adhesion modulator, induces apoptosis of rat Leydig cells in vitro

Galectin-1 (Gal-1), a beta galactoside-binding lectin, is involved in multiple biological functions, such as cell adhesion, apoptosis, and metastasis. On the basis of its ability to interact with extracellular matrix (ECM) glycoproteins, we investigated the Gal-1 effect on Leydig cells, which express and are influenced by ECM proteins. In this study, Gal-1 was identified in Leydig cell cultures by immunofluorescence. To gain insight into its biological role, Gal-1 was added to purified rat Leydig cells, under both basal and human chorionic gonadotrophin-stimulated conditions. Substantial morphological changes were observed, and cell viability showed an 80% decrease after 24 h culture. As a functional consequence of Gal-1 addition, testosterone production was reduced in a dose-dependent fashion, reaching a minimum of 26% after 24 h compared with basal values. cAMP showed a similar variation after 3 h. Assessment of DNA hypodiploidy and caspase activity determinations indicated that the reduction in viability and in steroidogenesis was caused by apoptosis induced by Gal-1. Besides, addition of Gal-1 caused Leydig cell detachment. Presence of laminin-1 or lactose prevented the effect of Gal-1, suggesting that the carbohydrate recognition domain is involved in inducing apoptosis. These findings demonstrate a novel mechanism, based on Gal-1 and laminin-1 interaction, which could help us better understand the molecular basis of Leydig cell function and survival control.

Effect of 5-azacytidine and galectin-1 on growth and differentiation of the human b lymphoma cell line bl36

Background5-AzaCytidine (AzaC) is a DNA demethylating drugs that has been shown to inhibit cell growth and to induce apoptosis in certain cancer cells. Induced expression of the galectin1 (Gal1) protein, a galactoside-binding protein distributed widely in immune cells, has been described in cultured hepatoma-derived cells treated with AzaC and this event may have a role in the effect of the drug. According to this hypothesis, we investigated the effect of AzaC and Gal1 on human lymphoid B cells phenotype.MethodsThe effect of AzaC and Gal1 on cell growth and phenotype was determined on the Burkitt lymphoma cell line BL36. An immunocytochemical analysis for detection of Gal1 protein expression was performed in AzaC-treated cells. To investigate the direct effects of Gal1, recombinant Gal1 was added to cells.ResultsTreatment of lymphoid B cells with AzaC results in: i) a decrease in cell growth with an arrest of the cell cycle at G0/G1 phase, ii) phenotypic changes consistent with a differentiated phenotype, and iii) the expression of p16, a tumor-suppressor gene whose expression was dependent of its promoter demethylation, and of Gal1. A targeting of Gal 1 to the plasma membrane follows its cytosolic expression. To determine which of the effects of AzaC might be secondary to the induction of Gal1, recombinant Gal1 was added to BL36 cells. Treated cells displayed growth inhibition and phenotypic changes consistent with a commitment toward differentiation.ConclusionsAltered cell growth and expression of the cell surface plasma cell antigen, CD138 are detectable in BL36 cells treated by AzaC as well as by Gal1. It seems that AzaC-induced Gal1 expression and consequent binding of Gal1 on its cell membrane receptor may be, in part, involved in AzaC-induced plasmacytic differentiation.

SCFMet30-mediated control of the transcriptional activator Met4 is required for the G1-S transition

Progression through the cell cycle requires the coordination of basal metabolism with the cell cycle and growth machinery. Repression of the sulfur gene network is mediated by the ubiquitin ligase SCF(Met30), which targets the transcription factor Met4p for degradation. Met30p is an essential protein in yeast. We have found that a met4Deltamet30Delta double mutant is viable, suggesting that the essential function of Met30p is to control Met4p. In support of this hypothesis, a Met4p mutant unable to activate transcription does not cause inviability in a met30Delta strain. Also, overexpression of an unregulated Met4p mutant is lethal in wild-type cells. Under non-permissive conditions, conditional met30Delta strains arrest as large, unbudded cells with 1N DNA content, at or shortly after the pheromone arrest point. met30Delta conditional mutants fail to accumulate CLN1 and CLN2, but not CLN3 mRNAs, even when CLN1 and CLN2 are expressed from strong heterologous promoters. One or more genes under the regulation of Met4p may delay the progression from G(1) into S phase through specific regulation of critical G(1) phase mRNAs.

Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells.

Galectin-1 (gal-1) a member of the mammalian beta-galactoside-binding proteins recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. In the present work, gal-1 has been identified to be a ligand for the CD3-complex as well as for CD2 as detected by affinity chromatography of Jurkat T-cell lysates on gal-1 agarose and by binding of the biotinylated lectin to CD3 and CD2 immunoprecipitates on blots. In CD45(+)Jurkat E6.1 cells, the lectin stimulates a sustained increase in the intracytoplasmic calcium concentration ([Ca(2+)](i)) consisting of both the release of calcium from intracellular stores and the calcium influx from the extracellular space. This effect of gal-1 on [Ca(2+)](i)is completely inhibited by lactose at 10 mM and was absent in CD45(-)Jurkat J45.01 cells. Preincubation of Jurkat E6.1 cells with cholera toxin or with the protein tyrosine kinase inhibitor herbimycin A reduced the gal-1 induced calcium response whereas the increase in [Ca(2+)](i)stimulated by CD2 or CD3 monoclonal antibodies (mAbs) was completely inhibited. Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on [Ca(2+)](i). Membrane depolarization with gramicidin or by a high-potassium buffer was without effects on the lectin-mediated calcium release from intracellular stores but inhibited the gal-1 induced receptor-operated calcium influx. In Jurkat E6.1 cells the lectin stimulates the transient generation of inositol-1,4,5-trisphosphate and the tyrosine phosphorylation of phospholipase Cgamma1. The results suggest that the ligation of CD2 and CD3 by gal-1 induces early events in T-cell activation comparable with that elicited by CD2 or CD3 mAbs.