The loss of DNA polymerase epsilon accessory subunits POLE3-POLE4 leads to BRCA1-independent PARP inhibitor sensitivity.

The clinical success of PARP1/2 inhibitors (PARPi) prompts the expansion of their applicability beyond homologous recombination deficiency. Here, we demonstrate that the loss of the accessory subunits of DNA polymerase epsilon, POLE3 and POLE4, sensitizes cells to PARPi. We show that the sensitivity of POLE4 knockouts is not due to compromised response to DNA damage or homologous recombination deficiency. Instead, POLE4 loss affects replication speed leading to the accumulation of single-stranded DNA gaps behind replication forks upon PARPi treatment, due to impaired post-replicative repair. POLE4 knockouts elicit elevated replication stress signaling involving ATR and DNA-PK. We find POLE4 to act parallel to BRCA1 in inducing sensitivity to PARPi and counteracts acquired resistance associated with restoration of homologous recombination. Altogether, our findings establish POLE4 as a promising target to improve PARPi driven therapies and hamper acquired PARPi resistance.

Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling.

PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling.

Polyploid Adipose Stem Cells Shift the Balance of IGF1/IGFBP2 to Promote the Growth of Breast Cancer.

Background: The close proximity of adipose tissue and mammary epithelium predispose involvement of adipose cells in breast cancer development. Adipose-tissue stem cells (ASCs) contribute to tumor stroma and promote growth of cancer cells. In our previous study, we have shown that murine ASCs, which undergo polyploidization during their prolonged in vitro culturing, enhanced the proliferation of 4T1 murine breast cancer cells in IGF1 dependent manner. Aims: In the present study, our aim was to clarify the regulation of ASC-derived IGF1. Methods: 4T1 murine breast carcinoma cells were co-transplanted with visceral fat-derived ASCs (vASC) or with the polyploid ASC.B6 cell line into female BALB/c mice and tumor growth and lung metastasis were monitored. The conditioned media of vASCs and ASC.B6 cells were subjected to LC-MS/MS analysis and the production of IGFBP2 was verified by Western blotting. The regulatory effect was examined by adding recombinant IGFBP2 to the co-culture of ASC.B6 and 4T1. Akt/protein kinase B (PKB) activation was detected by Western blotting. Results: Polyploid ASCs promoted the tumor growth and metastasis more potently than vASCs with normal karyotype. vASCs produced the IGF1 regulator IGFBP2, which inhibited proliferation of 4T1 cells. Downregulation of IGFBP2 by polyploidization of ASCs and enhanced secretion of IGF1 allowed survival signaling in 4T1 cells, leading to Akt phosphorylation. Conclusions: Our results implicate that ASCs in the tumor microenvironment actively regulate the growth of breast cancer cells through the IGF/IGFBP system.

TMEM203 is a binding partner and regulator of STING-mediated inflammatory signaling in macrophages.

Regulation of IFN signaling is critical in host recognition and response to pathogens while its dysregulation underlies the pathogenesis of several chronic diseases. STimulator of IFN Genes (STING) has been identified as a critical mediator of IFN inducing innate immune pathways, but little is known about direct coregulators of this protein. We report here that TMEM203, a conserved putative transmembrane protein, is an intracellular regulator of STING-mediated signaling. We show that TMEM203 interacts, functionally cooperates, and comigrates with STING following cell stimulation, which in turn leads to the activation of the kinase TBK1, and the IRF3 transcription factor. This induces target genes in macrophages, including IFN-ß. Using Tmem203 knockout bone marrow-derived macrophages and transient knockdown of TMEM203 in human monocyte-derived macrophages, we show that TMEM203 protein is required for cGAMP-induced STING activation. Unlike STING, TMEM203 mRNA levels are elevated in T cells from patients with systemic lupus erythematosus, a disease characterized by the overexpression of type I interferons. Moreover, TMEM203 mRNA levels are associated with disease activity, as assessed by serum levels of the complement protein C3. Identification of TMEM203 sheds light into the control of STING-mediated innate immune responses, providing a potential novel mechanism for therapeutic interventions in STING-associated inflammatory diseases.

Increased insulin-like growth factor 1 production by polyploid adipose stem cells promotes growth of breast cancer cells.

BACKGROUND: Adipose-tissue stem cells (ASCs) are subject of intensive research since their successful use in regenerative therapy. The drawback of ASCs is that they may serve as stroma for cancer cells and assist tumor progression. It is disquieting that ASCs frequently undergo genetic and epigenetic changes during their in vitro propagation. In this study, we describe the polyploidization of murine ASCs and the accompanying phenotypical, gene expressional and functional changes under long term culturing. METHODS: ASCs were isolated from visceral fat of C57BL/6 J mice, and cultured in vitro for prolonged time. The phenotypical changes were followed by microscopy and flow cytometry. Gene expressional changes were determined by differential transcriptome analysis and changes in protein expression were shown by Western blotting. The tumor growth promoting effect of ASCs was examined by co-culturing them with 4 T1 murine breast cancer cells. RESULTS: After five passages, the proliferation of ASCs decreases and cells enter a senescence-like state, from which a proportion of cells escape by polyploidization. The resulting ASC line is susceptible to adipogenic, osteogenic and chondrogenic differentiation, and expresses the stem cell markers CD29 and Sca-1 on an upregulated level. Differential transcriptome analysis of ASCs with normal and polyploid karyotype shows altered expression of genes that are involved in regulation of cancer, cellular growth and proliferation. We verified the increased expression of Klf4 and loss of Nestin on protein level. We found that elevated production of insulin-like growth factor 1 by polyploid ASCs rendered them more potent in tumor growth promotion in vitro. CONCLUSIONS: Our model indicates how ASCs with altered genetic background may support tumor progression.

Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells.

Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

Target-specific NMR detection of protein-ligand interactions with antibody-relayed 15N-group selective STD.

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein-ligand interactions is a key element. 1H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.

Galectin-1 is a local but not systemic immunomodulatory factor in mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have powerful immunosuppressive activity. This function of MSCs is attributed to plethora of the expressed immunosuppressive factors, such as galectin-1 (Gal-1), a pleiotropic lectin with robust anti-inflammatory effect. Nevertheless, whether Gal-1 renders or contributes to the immunosuppressive effect of MSCs has not been clearly established. Therefore, this question was the focus of a complex study. METHODS: MSCs were isolated from bone marrows of wild-type and Gal-1 knockout mice and their in vitro anti-proliferative and apoptosis-inducing effects on activated T cells were examined. The in vivo immunosuppressive activity was tested in murine models of type I diabetes and delayed-type hypersensitivity. RESULTS: Both Gal-1-expressing and -deficient MSCs inhibited T-cell proliferation. Inhibition of T-cell proliferation by MSCs was mediated by nitric oxide but not PD-L1 or Gal-1. In contrast, MSC-derived Gal-1 triggered apoptosis in activated T cells that were directly coupled to MSCs, representing a low proportion of the T-cell population. Furthermore, absence of Gal-1 in MSCs did not affect their in vivo immunosuppressive effect. CONCLUSIONS: These results serve as evidence that Gal-1 does not play a role in the systemic immunosuppressive effect of MSCs. However, a local contribution of Gal-1 to modulation of T-cell response by direct cell-to-cell interaction cannot be excluded. Notably, this study serves a good model to understand how the specificity of a pleiotropic protein depends on the type and localization of the producing effector cell and its target.

Novel real-time cell analysis platform for the dynamic monitoring of ionizing radiation effects on human tumor cell lines and primary fibroblasts.

Translational research in radiation oncology is important for the detection of adverse radiation effects, cellular responses, and radiation modifications, and may help to improve the outcome of radiation therapy in patients with cancer. The present study aimed to optimize and validate a real­time label­free assay for the dynamic monitoring of cellular responses to ionizing radiation. The xCELLigence system is an impedance­based platform that provides continuous information on alterations in cell size, shape, adhesion, proliferation, and survival. In the present study, various malignant human primary fibroblast cells (U251, GBM2, MCF7, A549, HT­29) were exposed to 0, 5 and 10 Gy of Cobalt60 radiation. As well as the xCELLigence system, cell survival and proliferation was evaluated using the following conventional end­point cell­based methods: Clonogenic, MTS, and lactate dehydrogenase assays, and apoptosis was detected by fluorescence­activated cell sorting. The effects of ionizing radiation were detected for each cell line using impedance monitoring. The real­time data correlated with the colony forming assay results. At low cell densities (1,000­2,000 cells/well) the impedance­based method was more accurate at monitoring dose­dependent changes in the malignant human primary fibroblast cell lines, as compared with the end­point assays. The results of the present study demonstrated that the xCELLigence system may be a reliable and rapid diagnostic method for the monitoring of dynamic cell behavior following radiation. In addition, the xCELLigence system may be used to investigate the cellular mechanisms underlying the radiation response, as well as the time­dependent effects of radiation on cell proliferation and viability.

Licensing by Inflammatory Cytokines Abolishes Heterogeneity of Immunosuppressive Function of Mesenchymal Stem Cell Population.

When mesenchymal stem cells (MSCs) are used for therapy of immunological pathologies, they get into an inflammatory environment, altering the effectiveness of the treatment. To establish the impact of environmental inflammatory factors on MSCs' immunofunction in the mirror of intrinsic heterogeneity of mouse MSC population, individual MSC clones were generated and characterized. Adipogenic but not osteogenic differentiation and pro-angiogenic activity of five independent MSC cell lines were similar. Regarding osteogenic differentiation, clones MSC3 and MSC6 exhibited poorer capacity than MSC2, MSC4, and MSC5. To study the immunosuppressive heterogeneity, in vitro and in vivo experiments have been carried out using T-cell proliferation assay and delayed-type hypersensitivity (DTH) response, respectively. A remarkable difference was found between the clones in their ability to inhibit T-cell proliferation in the following order: MSC2≥MSC5>MSC4>MSC3 >> MSC6. Nevertheless, the differences between the immunosuppressive activities of the individual clones disappeared on pretreatment of the cells with pro-inflammatory cytokines, a procedure called licensing. Stimulation of all clones with IFN-γ and TNF-α resulted in elevation of their inhibitory capability to a similar level. Nitric oxide (NO) and prostaglandin E2 (PGE2) were identified as major mediators of immunofunction of the MSC clones. The earlier findings were also supported by in vivo results. Without licensing, MSC2 inhibited DTH response, while MSC6 did not affect DTH response. In contrast, prestimulation of MSC6 with inflammatory cytokines resulted in strong suppression by this clone as well. Here, we have showed that MSC population is functionally heterogeneous in terms of immunosuppressive function; however, this variability is largely reduced under pro-inflammatory conditions.

Novel role for galectin-1 in T-cells under physiological and pathological conditions.

Secreted, extracellular galectin-1 (exGal-1) but not intracellular Gal-1 (inGal-1) has been described as a strong immunosuppressive protein due to its major activity of inducing apoptosis of activated T-cells. It has previously been reported that T-cells express Gal-1 upon activation, however its participation in T-cell functions has remained largely elusive. To determine function of Gal-1 expressed by activated T-cells we have carried out a series of experiments. We have shown that Gal-1, expressed in Gal-1-transgenic Jurkat cells or in activated T-cells, remained intracellularly indicating that Gal-1-induced T-cell death was not a result of an autocrine effect of the de novo expressed Gal-1. Rather, a particular consequence of the inGal-1 expression was that T-cells became more sensitive to exGal-1 added either as a soluble protein or bound to the surface of a Gal-1-secreting effector cell. This was also verified when the susceptibility of activated T-cells from wild type or Gal-1 knockout mice to Gal-1-induced apoptosis were compared. Murine T-cells expressing Gal-1 were more sensitive to the cytotoxicity of the exGal-1 than their Gal-1 knockout counterparts. We also conducted a study with activated T-cells from patients with systemic lupus erythematosus (SLE), a disease in which dysregulated T-cell apoptosis has been well described. SLE T-cells expressed lower amounts of Gal-1 than healthy T-cells and were less sensitive to exGal-1. These results suggested a novel role of inGal-1 in T-cells as a regulator of T-cell response to exGal-1, and its likely contribution to the mechanism in T-cell apoptosis deficiency in lupus.

GM1 controlled lateral segregation of tyrosine kinase Lck predispose T-cells to cell-derived galectin-1-induced apoptosis.

One prominent immunoregulatory function of galectin-1 (Gal-1), a ß-galactoside binding mammalian lectin, is induction of apoptosis in activated T-cells by a process depending on the activity of Src family tyrosine kinase, Lck. Although the requirement for Lck in Gal-1 induced T-cell death and the ability of Gal-1 to affect the membrane localization of extracellular Gal-1-binding proteins have been well documented, the consequence of the complex and related reorganization of extra- and intracellular signaling components upon Gal-1 treatment of T-cells has not yet been revealed. Therefore, we have analyzed the plasma membrane movement of Lck upon Gal-1 triggered signaling, and the significance of this event in Gal-1 induced T-cell death. Non-receptor tyrosine kinase, Lck primarily localized in the synapse of tumor cell-T-cell during 15 min of the established direct cell contact. Later, after 30 min, a lateral segregation of Lck from the cell synapse was observed. The migration of Lck to the opposite of the cell contact apparently depended on the expression and cell surface presentation of Gal-1 on the effector (tumor) cells and was accompanied by phosphorylation on the negative regulatory tyrosine residue, Tyr505. Receptor tyrosine phosphatase, CD45 played crucial role in this event since CD45 deficiency or inhibition of its phosphatase activity resulted in the failure of Lck membrane movement. Level of the Gal-1-binding glycolipid GM1 ganglioside also essentially regulated Lck localization. Segregation of Lck and Gal-1 induced apoptosis was diminished in T-cells with low GM1 expression compared to T-cells with high GM1. Our results show that spatial regulation of Lck by CD45 and GM1 ganglioside determines the outcome of apoptotic response to Gal-1 and this local regulation may occur only upon intimate effector (Gal-1 expressing) cell-T-cell attachment.

Identification of galectin-1 as a critical factor in function of mouse mesenchymal stromal cell-mediated tumor promotion.

Bone marrow derived mesenchymal stromal cells (MSCs) have recently been implicated as one source of the tumor-associated stroma, which plays essential role in regulating tumor progression. In spite of the intensive research, the individual factors in MSCs controlling tumor progression have not been adequately defined. In the present study we have examined the role of galectin-1 (Gal-1), a protein highly expressed in tumors with poor prognosis, in MSCs in the course of tumor development. Co-transplantation of wild type MSCs with 4T1 mouse breast carcinoma cells enhances the incidence of palpable tumors, growth, vascularization and metastasis. It also reduces survival compared to animals treated with tumor cells alone or in combination with Gal-1 knockout MSCs. In vitro studies show that the absence of Gal-1 in MSCs does not affect the number of migrating MSCs toward the tumor cells, which is supported by the in vivo migration of intravenously injected MSCs into the tumor. Moreover, differentiation of endothelial cells into blood vessel-like structures strongly depends on the expression of Gal-1 in MSCs. Vital role of Gal-1 in MSCs has been further verified in Gal-1 knockout mice. By administering B16F10 melanoma cells into Gal-1 deficient animals, tumor growth is highly reduced compared to wild type animals. Nevertheless, co-injection of wild type but not Gal-1 deficient MSCs results in dramatic tumor growth and development.These results confirm that galectin-1 is one of the critical factors in MSCs regulating tumor progression.

Positional identity of murine mesenchymal stem cells resident in different organs is determined in the postsegmentation mesoderm.

Although mesenchymal stem cells (MSCs) of distinct tissue origin have a large number of similarities and differences, it has not been determined so far whether tissue-resident MSCs are the progenies of one ancestor cell lineage or the results of parallel cell developmental events. Here we compared the expression levels of 177 genes in murine MSCs derived from adult and juvenile bone marrow and adult adipose tissue, as well as juvenile spleen, thymus, and aorta wall by quantitative real-time polymerase chain reaction and the results were partially validated at protein level. All MSC lines uniformly expressed a large set of genes including well-known mesenchymal markers, such as α-smooth muscle actin, collagen type I α-chain, GATA6, Mohawk, and vimentin. In contrast, pluripotency genes and the early mesodermal marker T-gene were not expressed. On the other hand, different MSC lines consistently expressed distinct patterns of Hox genes determining the positional identity of a given cell population. Moreover, MSCs of different origin expressed a few other transcription factors also reflecting their topological identity and so the body segment or organ to which they normally contributed in vivo: (1) thymus-derived cells specifically expressed Tbx5 and Pitx2; (2) spleen-derived MSCs were characterized with Tlx1 and Nkx2.5; (3) Pitx1 designated femoral bone marrow cells and (4) En2 appeared in aorta wall-derived MSCs. Thus, MSCs exhibited topographic identity and memory even after long-term cultivation in vitro. On the basis of these results, we suggest that postnatal MSCs isolated from different anatomical sites descend from precursor cells developing in the postsegmentation mesoderm.

How does it act when soluble? Critical evaluation of mechanism of galectin-1 induced T-cell apoptosis.

Galectin-1 (Gal-1), a mammalian lectin induces apoptosis of T lymphocytes. Contradictory data have resulted in confusing knowledge regarding mechanism of Gal-1 induced T-cell apoptosis. In this paper we aimed to resolve this controversy by comparing cell death induced by low (1.8 µM, lowGal-1) and high (18 µM, highGal-1) concentration of soluble Gal-1. We show that lowGal-1 and highGal-1 trigger phosphatidylserine exposure, generation of rafts and mitochondrial membrane depolarization. In contrast, lowGal-1 but not highGal-1 is dependent on the presence of p56lck and ZAP70 and activates caspase cascade. The results allow the conclusion that the cell-death mechanism strictly depends on the concentration of Gal-1.

Mechanism of tumor cell-induced T-cell apoptosis mediated by galectin-1.

Galectin-1 (Gal-1) has been implicated in tumor progression partly via the induction of T-cell apoptosis. However the mechanism of Gal-1 induced T-cell death was mostly studied using recombinant, soluble Gal-1 producing controversial results. To explore the true mechanism of Gal-1 and hence tumor cell-induced T-cell death, we applied co-cultures of tumor cells and T-cells thus avoiding artificial circumstances generated using recombinant protein. T-cells died when co-cultured with Gal-1-expressing but survived with Gal-1 non-expressing tumor cells. Removing tumor cell surface Gal-1 or knocking down Gal-1 expression resulted in diminution of T-cell apoptosis. Gal-1 transgenic or soluble Gal-1 treated HeLa cells became cytotoxic. Stimulation of apoptosis required interaction between the tumor and T-cells, presence of p56lck and ZAP70, decrease of mitochondrial membrane potential and caspase activation. Hence tumor cell-derived Gal-1 might efficiently contribute to tumor self-defense. Moreover this system resolves the discrepancies obtained using recombinant Gal-1 in T-cell apoptosis studies.

Galectin-1-asialofetuin interaction is inhibited by peptides containing the tyr-xxx-tyr motif acting on the glycoprotein.

Galectin-1 (Gal-1), a ubiquitous beta-galactoside-binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal-1 depend on its affinity for beta-galactoside-containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr-Xxx-Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal-1-asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr-Xxx-Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr-Xxx-Tyr peptides studied do not bind to Gal-1, whereas their binding to ASF is clearly detected. (15)N,(1)H HSQC titrations with (15)N-labeled Gal-1 confirm the absence of any peptide-Gal-1 interaction. These data indicate that the Tyr-Xxx-Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.

A novel anti-inflammatory function of human galectin-1: inhibition of hematopoietic progenitor cell mobilization.

OBJECTIVE: The immunosuppressive and anti-inflammatory activity of mammalian galectin-1 (Gal-1) has been well established in experimental in vivo animal models and in vitro studies. Since the proliferation and migration of leukocytes represent a necessary and important step in response to the inflammatory insult, we have investigated whether Gal-1 affects the mobilization of hematopoietic progenitor cells (HPC) induced by cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). METHODS: Bone marrow HPCs were mobilized with CY/G-CSF or CY/G-CSF plus human recombinant Gal-1 in BDF1 mice. Bone marrow (BM) and blood cells were taken at different time points and analyzed for their in vivo repopulating ability in lethally irradiated syngeneic animals. The number of myeloid progenitor cells in BM and blood samples was determined by colony-forming cell assay. Expression of surface markers (Sca-1, CD3epsilon, CD45R/B220, Ter-119, GR-1, and CD11b) on nucleated marrow cells was measured by flow cytometry. The lymphocytes, granulocytes, and monocytes in blood samples were counted after Giemsa staining. RESULTS: Gal-1 dramatically inhibited CY/G-CSF-induced HPC migration to the periphery as well as decreased peripheral neutrophilia and monocytosis in a dose- and time-dependent manner. In contrast, Gal-1 itself stimulated HPC expansion and accumulation within the BM. The presence of the lectin for inhibition of HPC mobilization was essential during the second half of the treatment. Moreover, Gal-1 inhbited transendothelial migration of BM-derived HPCs in response to SDF-1 in vitro. CONCLUSION: Gal-1 blocked BM progenitor cell migration induced by CY/G-CSF treatment, indicating a novel anti-inflammatory function of the lectin. We suggest that the inhibition of HPC mobilization occurs mainly via obstructing the transendothelial migration of BM-derived cells including primitive hematopoietic and committed myeloid progenitor cells and mature granulocytes and monocytes.

Acid sphingomyelinase mediated release of ceramide is essential to trigger the mitochondrial pathway of apoptosis by galectin-1.

The mechanism of apoptosis induced by human galectin-1, a mammalian beta-galactoside-binding protein with a remarkable cytotoxic effect on activated peripheral T cells and tumor T cell lines has been extensively investigated in this study. Here we first show that galectin-1 initiate the acid sphingomyelinase mediated release of ceramide and this event is critical in the further steps. Elevation of ceramide level coincides with exposure of phosphatidylserine on the outer cell membrane. The downstream events, decrease of Bcl-2 protein amount, depolarization of the mitochondria and activation of the caspase 9 and caspase 3 depend on production of ceramide. All downstream steps, including production of ceramide, require the generation of membrane rafts and the presence of two tyrosine kinases, p56(lck) and ZAP70. Based on our findings we suggest a model of the mechanism of galectin-1 triggered cell death.

Biphasic effect of recombinant galectin-1 on the growth and death of early hematopoietic cells.

Galectin-1 is a member of the family of beta-galactoside binding animal lectins, galectins. Its presence in the bone marrow has been detected; however, its role in the regulation of hematopoiesis is unknown. In the present study, we have evaluated the effect of recombinant human galectin-1 on the proliferation and survival of murine and human hematopoietic stem and progenitor cells. We show that low amount of galectin-1 (10 ng/ml) increases the formation of granulocyte-macrophage and erythroid colonies and the frequencies of day-7 cobblestone area-forming cells on a lactose-inhibitable fashion. In contrast, high amount of galectin-1 (10 microg/ml) dramatically reduces the growth of the committed blood-forming progenitor cells as well as the much younger, lineage-negative hematopoietic cells (day-28 to -35 cobblestone area-forming cells). This inhibition is not blocked by lactose and, therefore, is largely independent of the beta-galactoside-binding site of the lectin. Furthermore, assays to detect apoptosis render it likely that the high amount of galectin-1 acts as a classical proapoptotic factor for the premature hematopoietic cells.