Lewis X Research Articles

LNFPIII/LeX-Stimulated Macrophages Activate Natural Killer Cells via CD40-CD40L Interaction

Lacto-N-fucopentaose III (LNFPIII) is a human milk sugar containing the biologically active Lewis X (LeX) trisaccharide. LNFPIII/LeX is also expressed by immunosuppressive helminth parasites, by bacteria, and on a number of tumor/cancer cells. In this report, we first demonstrate that LNFPIII activates macrophages in vitro as indicated by upregulation of Gr-1 expression on F4/80(+) cells. Further, we investigated the effect of LNFPIII-activated macrophages on NK cell activity. We found that LNFPIII-stimulated F4/80(+) cells were able to activate NK cells, inducing upregulation of CD69 expression and gamma interferon (IFN-gamma) production. The experiments show that NK cell activation is macrophage dependent, since NK cells alone did not secrete IFN-gamma in response to LNFPIII. Furthermore, we found that activation of NK cells by glycan-stimulated macrophages required cell-cell contact. As part of the cell-cell contact mechanism, we determined that CD40-CD40L interaction was critical for IFN-gamma secretion by NK cells, as the addition of anti-CD40L antibodies to the coculture blocked IFN-gamma production. We also demonstrated that LNFPIII-stimulated macrophages secrete prostaglandin E(2), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-alpha) but a very low level of IL-12. Interestingly, addition of anti-TNF-alpha, anti-IL-10, or anti-IL-12 monoclonal antibodies did not significantly alter NK cell activity. Our data show that these soluble mediators are not critical for LNFPIII-stimulated macrophage activation of NK cells and provide further evidence for the importance of cell-cell contact and CD40-CD40L interactions between macrophages and NK cells.

Targeted ultrasound contrast agents: In vitro assessment of endothelial dysfunction and multi‐targeting to ICAM‐1 and sialyl Lewisx

An ultrasound-based molecular imaging technique capable of detecting endothelial cell markers of inflammation may allow early, non-invasive assessment of vascular disease. Clinical application of targeted, acoustically-active microbubbles requires optimization of microbubble-endothelial adhesion strength to maximize image signal-to-noise ratio, as well as the ability to discern the degree of inflammation along a continuum of dysfunction. Accordingly, we hypothesized that adhesion of intercellular adhesion molecule-1 (ICAM-1)-targeted microbubbles is dependent on the degree of endothelial inflammation, and that microbubbles multi-targeted to both ICAM-1 (via anti-ICAM-1 antibodies) and selectins (via sialyl Lewisx) demonstrate greater adhesion strength than microbubbles targeted to either inflammatory marker alone. In a radial flow chamber, microbubbles were perfused across endothelial cells activated with interleukin-1beta to four different levels of inflammation, as assessed by quantitative ICAM-1 expression. ICAM-1-targeted microbubble adhesion strength increased with increasing degree of inflammation, with a relationship that was both positive and linear (r > 0.99). Microbubble adhesion strength was significantly higher for the multi-targeted microbubbles than either of the single-targeted microbubbles. These data thus demonstrate that multi-targeting of contrast microbubbles may offer improved adhesion characteristics, allowing for greater sensitivity to inflammation. Furthermore, the adhesion strength of targeted microbubbles is linearly dependent on the degree of inflammation, suggesting that targeted ultrasound imaging may offer differentiation between various degrees of endothelial dysfunction, and thus detect not only the presence, but also the severity of inflammatory disease processes.

Conformational Dynamics of Sialyl Lewisx in Aqueous Solution and Its Interaction with SelectinE. A Study by Molecular Dynamics

Three dimensional structures of sialyl Lewisx (SLex) in aqueous solution and bound to selectinE are described based on an exhaustive conformational analysis and several long molecular dynamics simulations using different glycosidic regions as starting conformations. It appears from this study that when the oligosaccharide is free in solution the NeuNAca(2–3)Gal segment favors glycosidic conformation in three different regions in the plane with propensity of populations in the ratio 1:8:1. Each one of these structures is characteristically stabilized by specific hydrogen bonding interaction between NeuNAc and Gal. On the other hand, the Gal-GlcNAc-Fuc segment can exist in four different conformational states. Based on the topology of SLex we are able to predict that out of all the allowed conformations in solution only two of these structures possess a geometry that would fit without steric clashes into the binding location of selectinE. In both of these binding modes, segment Gal-GlcNAc-Fuc adopts a unique conformation. The only difference between the two SLex conformers that can successfully bind to selectinE is given by two possible regions in glycosidic space in the fragment NeuNAca(2–3)Gal. A large conformational departure from the crystallographic data is observed for two lysine residues at the binding site of selectinE. These two residues play an important role when SLex binds selectinE in aqueous solution. These findings help reconcile the X-ray data, in which these residues appear to be 1 nm away from SLex, with recent liquid NMR data reporting couplings between these protein residues and the sugar.

Introduction of Sda Carbohydrate Antigen in Gastrointestinal Cancer Cells Eliminates Selectin Ligands and Inhibits Metastasis

The Sd(a) blood group carbohydrate structure is expressed in the normal gastrointestinal mucosa. We reported previously that the expression of Sd(a) carbohydrate structures and beta1,4-N-acetylgalactosaminyltransferase (beta1,4GalNAcT) activity responsible for Sd(a) synthesis were remarkably decreased in cancer lesions of the gastrointestinal tract. In this study, we found that Sd(a) antigen was expressed mainly in chief cells of normal stomach but not in cancer tissue by immunohistologic staining. In separated gastric mucosal cells, the Sd(a) glycolipids and beta1,4GalNAcT activity were concentrated in a fraction that contained chief cells as a major population. We cloned the cDNA encoding the glycosyltransferase that catalyzes the synthesis of Sd(a) (Sd(a)-beta1,4GalNAcT). Introduction of this cloned cDNA into KATO III gastric or HT29 colonic cancer cell lines, which originally expressed the E-selectin ligands, sialyl Lewis(x) and sialyl Lewis(a), resulted in a marked increase in cell-surface expression of Sd(a) along with the concomitant total loss of both sialyl Lewis(x) and sialyl Lewis(a). Both KATO III and HT29 cells transfected with the Sd(a)-beta1,4GalNAcT gene showed significantly decreased adhesion to activated human umbilical vein endothelial cells when compared with mock-transfected cells. Sd(a) determinants showed no direct binding to Siglec-3, -5, -7, and -9. These Sd(a)-beta1,4GalNAcT-transfected cells showed strikingly reduced metastatic potential in vivo when compared with mock-transfected cells. In summary, forced expression of Sd(a) carbohydrate determinant caused remarkable elimination of carbohydrate ligands for selectin and reduced metastasis of human gastrointestinal tract cancer cells.

Affinity and Kinetics of Sialyl Lewis-X and Core-2 Based Oligosaccharides Binding to L- and P-Selectin

Soluble oligosaccharide mimetics of natural selectin ligands act as competitive inhibitors of leukocyte adhesion in models of inflammation. We quantified the binding of simple oligosaccharides based on sialyl Lewis-X (sLe(X)) and complex molecules with the core-2 structure to L- and P-selectin, under both static and fluid flow conditions. Isolated human neutrophils were employed to mimic the physiological valency of selectins and selectin ligands. Surface plasmon resonance studies quantified binding kinetics. We observed the following: (i) The functional group at the anomeric position of carbohydrates plays an important role during selectin recognition, since sLe(X) and sialyl Lewis-a (sLe(a)) were approximately 5-7-fold poorer inhibitors of L-selectin mediated cell adhesion compared to their methyl glycosides. (ii) Despite their homology to physiological glycans, the putative carbohydrate epitopes of GlyCAM-1 and PSGL-1 bound selectins with low affinity comparable to that of sLe(X)-selectin interactions. Thus, besides the carbohydrate portion, the protein core of GlyCAM-1 or the presentation of carbohydrates in clusters on this glycoprotein may contribute to selectin recognition. (iii) A compound Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(GalNAcbeta1,3)GalNAcalpha-OMe was identified which blocked L- and P-selectin binding at 30-100-fold lower doses than sLe(X). (iv) Surface plasmon resonance experiments determined that an sLe(X) analogue (TBC1269) competitively inhibited, via steric/allosteric mechanisms, the binding of two anti-P-selectin function blocking antibodies that recognized different epitopes of P-selectin. (v) TBC1269 bound P-selectin via both calcium-dependent and -independent mechanisms, with K(D) of approximately 111.4 microM. The measured on- and off-rates were high (k(off) > 3 s(-)(1), k(on) > 27,000 M(-)(1) s(-)(1)). Similar binding kinetics are expected for sLe(X)-selectin interactions. Taken together, our study provides new insight into the kinetics and mechanisms of carbohydrate interaction with selectins.

An Adenoid Cystic Carcinoma Cell Line Possessing High Metastatic Activity has High NF-κB Activation in Response to TNF-α

Adenoid cystic carcinoma (AdCC)is characterized by frequent recurrence and distant metastasis. Although lung metastasis in AdCC is very common, the mechanism by which this occurs is uncertain. When five AdCC cell lines(ACCS, ACCT, ACCH, Acc-3, and Acc-M) were screened for metastatic ability by injecting tumor cells into nude mice via the tail vein, lung metastases were found in mice injected with Acc-M (15/16 mice) but not in mice injected with any of the other four cell lines (0/10 mice with each line). To determine why Acc-M metastasizes to the lung but the others do not, we examined the biological characteristics of Acc-M and compared them with those of the other lines.Nuclear factor-κB (NF-κB) may play a key role in malignant tumor behaviors such as invasion and metastasis. Thus, we examined these cell lines for response to tumor necrosis factor (TNF-α), one of the typical stimulators of NF-κB. Although treatment with TNF-α stimulated matrix metalloprotease 9 (MMP-9) expression in all cell lines, the response to TNF-α varied between cell lines; the greatest stimulation was observed in Acc-M. Acc-M expressed higher levels of TNF receptors (both TNF-R1 and TNF-R2) than did the other AdCC lines. Judging from inhibitor-κBα degradation and nuclear translocation and DNA binding by NF-κB, the degree of activation of NF-κB in response to TNF-α in Acc-M cell lines was very high compared to the other lines. Moreover, the ability of Acc-M cells to adhere to endothelial cells, which was greater than that of the other cell lines, was further enhanced by pretreatment with TNF-α. Acc-M cells also expressed higher levels of sialyl Lewisx than did the other AdCC cell lines. These findings suggest that lung metastasis is mediated by tumor-endothelial cell interaction, which is probably associated with the NF-κB activation pathway. Further experiments are required to identify the molecules that mediate both lung metastasis and NF-κB activation.

Rolling dynamics of a neutrophil with redistributed L-selectin

The most common white blood cell is the neutrophil, which slowly rolls along the walls of blood vessels due to the coordinated formation and breakage of chemical selectin–carbohydrate bonds. We show that L-selectin receptors are rapidly redistributed to form a cap at one end of the cell membrane during rolling via selectins or chemotactic stimulation. This topography significantly alters the adhesive dynamics as demonstrated by computer simulations of neutrophils rolling on a carbohydrate selectin-ligand substrate under flow. It was found that neutrophils with a redistributed L-selectin cap roll on sialyl Lewis-x with a quasi-periodic motion, as characterized by relatively low velocity intervals interspersed with regular jumps in the rolling velocity. On average, neutrophils with redistributed L-selectin rolled at a lower velocity when compared with cells having a uniform L-selectin distribution of equal average density. We speculate on the possible biological implications that these differences in adhesion dynamics will have during the inflammatory response.

Effect of surface modified liposomes on the aggregation of platelets and tumor cells

Metastasis is still the most serious reason for the high mortality of cancer patients. It is a complex process in which platelets play a crucial role. Several attempts have been performed to inhibit the metastatic process, some of these using modified liposomes. The aggregation behaviour of human platelets and HT29 colon carcinoma cells in the presence of liposomes with a modified surface has been investigated in the present study. Liposomes (PC/CH/DMPE) were unmodified, sterically stabilized by polyethylene glycol (PEG-DSPE), or equipped with the carbohydrate ligand sialyl Lewis(X) (conjugated to PEG-DMPE or DMPE as anchor) intended to specifically compete with ligands expressed by HT29 cells. We found in vitro that an addition of surface modified liposomes to human platelets in plasma caused an up to 2.9-fold increase in platelet aggregation. In addition, when HT29 tumor cells were mixed with platelets and surface modified liposomes, the number of tumor cells found in aggregates increased significantly from 8.3 % (only tumor cells) to 30.2 %. This result was supported by fluorescence micrographs demonstrating a strong association of platelets and liposomes around the tumor cells. In addition, a clear decrease in number and a change in the distribution of metastases after intravenous injection of HT29 cells in combination with liposomes was observed in vivo. While in control mice metastases in lung, liver and in intestine were prevailing, liposomal treatment resulted in a new localization of metastases in muscles. Taking together, the ability of surface modified liposomes to enhance aggregate formation of platelets and tumor cells has been demonstrated for the first time. The capability of these vesicles to interfere with the metastatic process might have implications for the use of such liposomes for therapeutic applications.

PD.161 Sialyl lewis-X expression confers endothelial binding capacity on circulating oral cancer cells

PD.161 Sialyl lewis-X expression confers endothelial binding capacity on circulating oral cancer cells

Suppressed transcription of genes for sulfate transporter, DTDST, as a major mechanism for induction of a carbohydrate tumor marker sialyl Lewisx in colon cancers

Suppressed transcription of genes for sulfate transporter, DTDST, as a major mechanism for induction of a carbohydrate tumor marker sialyl Lewisx in colon cancers

Design and Synthesis of Leukocyte Adhesion Inhibitors

The recognition of glycoprotein ligands by the selectins mediates leukocyte recruitment during an inflammatory response. Mimetics whose designs are based on the sialyl Lewis-x tetrasaccharide, common to these glycoproteins, have proven to be good inhibitors of leukocyte binding to the selectins. Some of the more recent approaches to the synthesis of these mimetics, including the use of polymeric and liposomal displays, multicomponent condensation reactions, and solid-phase peptide synthesis, are presented.

Shear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation of high-avidity beta2-integrin on neutrophils.

Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewis(x) ligands and activation of beta(2)-integrins that support neutrophil arrest by binding ICAM-1. We have previously reported that neutrophils rolling on E-selectin are sufficient for signaling cell arrest through beta(2)-integrin binding of ICAM-1 in a process dependent upon ligation of L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1). Unresolved are the spatial and temporal events that occur as E-selectin binds to human neutrophils and dynamically signals the transition from neutrophil rolling to arrest. Here we show that binding of E-selectin to sialyl Lewis(x) on L-selectin and PSGL-1 drives their colocalization into membrane caps at the trailing edge of neutrophils rolling on HUVECs and on an L-cell monolayer coexpressing E-selectin and ICAM-1. Likewise, binding of recombinant E-selectin to PMNs in suspension also elicited coclustering of L-selectin and PSGL-1 that was signaled via mitogen-activated protein kinase. Binding of recombinant E-selectin signaled activation of beta(2)-integrin to high-avidity clusters and elicited efficient neutrophil capture of beta(2)-integrin ligands in shear flow. Inhibition of p38 and p42/44 mitogen-activated protein kinase blocked the cocapping of L-selectin and PSGL-1 and the subsequent clustering of high-affinity beta(2)-integrin. Taken together, the data suggest that E-selectin is unique among selectins in its capacity for clustering sialylated ligands and transducing signals leading to neutrophil arrest in shear flow.

NG2-expressing cells in the subventricular zone are type C-like cells and contribute to interneuron generation in the postnatal hippocampus.

The subventricular zone (SVZ) is a source of neural progenitors throughout brain development. The identification and purification of these progenitors and the analysis of their lineage potential are fundamental issues for future brain repair therapies. We demonstrate that early postnatal NG2-expressing (NG2+) progenitor cells located in the SVZ self-renew in vitro and display phenotypic features of transit-amplifier type C–like multipotent cells. NG2+ cells in the SVZ are highly proliferative and express the epidermal growth factor receptor, the transcription factors Dlx, Mash1, and Olig2, and the Lewis X (LeX) antigen. We show that grafted early postnatal NG2+ cells generate hippocampal GABAergic interneurons that propagate action potentials and receive functional glutamatergic synaptic inputs. Our work identifies Dlx+/Mash1+/LeX+/NG2+/GFAP-negative cells of the SVZ as a new class of postnatal multipotent progenitor cells that may represent a specific cellular reservoir for renewal of postnatal and adult inhibitory interneurons in the hippocampus.

Study on relationship of fucosyltransferase gene types in breast cancer with metastasis and prognosis

To assess the association of five types of fucosyltransferase gene (Fuc-T), the important biosynthesis gene of sialylated carbohydrate antigens (Sialyl Lewis A, Sialyl Lewis X), with metastasis and prognosis of breast cancer. The real-time quantitative PCR of five-type Fuc-T III, IV, V, VI, VII genes was performed in 80 patients with breast cancer. The result showed that Fuc-TVII gene had higher gene copy compared to other type of Fuc-T in breast cancer. The grading of Fuc-TVII gene was related to lymph node metastasis and poor disease-free survival. Statistically difference was significant (P < 0.01). Fuc-T III, IV, V, VI gene were not significant relation to the metastasis and prognosis in 80 cases. These findings suggest that Fuc-TVII gene is a prognostic indicator of breast cancer, and it may be play an important role in the biosynthesis of sialylated carbohydrate antigens in breast cancer.

Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis.

Malignant transformation is associated with abnormal glycosylation, resulting in the synthesis and expression of altered carbohydrate determinants including sialyl Lewisa and sialyl Lewisx. The sialyl Lewisa and sialyl Lewisx determinants appear in the sera of patients with cancer, and are extensively utilized for serum diagnosis of cancers in Japan. Sialyl Lewisa and sialyl Lewisx are involved in selectin-mediated adhesion of cancer cells to vascular endothelium, and these determinants are thought to be closely associated with hematogenous metastasis of cancers. Recent progress in this area includes the following: 1. Substantial increases in solid clinical statistics that further confirm the contribution of these determinants in the progression of a wide variety of cancers; 2. Elucidation of the ligand specificity of the three family members of selectins and evaluation of the roles of these molecules in cancer cell adhesion; and 3. Advances in the study of the mechanism that leads to the enhanced expression of the sialyl Lewis(a/x) determinants in malignant cells. These recent results have confirmed that these determinants are not merely markers for cancers, but are functionally implicated in the malignant behavior of cancer cells. The results also suggested that the increase of these determinants in malignant cells is an inevitable consequence of the malignant transformation of cells. Considerable new knowledge has also been accumulated regarding the therapeutic implications for suppression of hematogenous metastasis targeting this cell adhesion system.

Synthesis of photoreactive neoglycolipid probes--instruments for studying membrane lectins

A method for the synthesis of photoaffinity neoglycolipid probes with a highly efficient carbene-generating diazocyclopentadien-2-ylcarbonyl (Dcp) label, which can be radioiodinated under standard oxidation conditions, was developed. The probes are intended for incorporation into the lipid bilayer. They are lipophilic glycoconjugates on the basis of an amphiphilic aglycone built up from a diacylglycerol and a polyethylene glycol spacer (with a polymerization degree of 9-16) bearing the Dcp label at the terminal unit. The location of the label in the aglycone provides the possibility of one-step preparation of a wide range of probes using various carbohydrate synthons. We have synthesized photoaffinity neoglycoconjugates containing the oligosaccharides: sialyl LewisX tetrasaccharide and A trisaccharide, which is specific to some tumor cells. A probe containing an inactive pentaol (aminodeoxyglucitol) was also synthesized to detect nonspecific binding. The Dcp label is bound to the probe molecule by ester bond; its lability under alkaline conditions facilitates the analysis of cross-linked products after photoaffinity labeling. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

Functional microdomains of glycolipids with partially fluorinated membrane anchors: impact on cell adhesion.

Functional microdomains of glycolipids were designed by mixing neoglycolipids with partially fluorinated alkyl (F-alkyl) chains and matrix lipids with alkyl chains. Fluorescence images of the mixed lipid monolayers at the air-water interface demonstrated that it is possible to control both size and distribution of the microdomains by means of the strong demixing of alkyl and F-alkyl membrane anchors, while the carbohydrate head groups seemed to play a rather minor role. These microdomains in monolayers could be transferred onto hydrophobized substrates and subjected to experiments in a dynamic flow chamber. The results obtained here clearly indicated that the dynamic adhesion of Chinese hamster ovarial cells expressing E-selectin (CHO-E cells) on a lipid monolayer containing microdomains of sialyl Lewisx (sLex) can be both enhanced and reduced by controlled demixing of ligands and matrices. Moreover, the same clusters of sLex could also be formed in giant lipid vesicles, which can be used as a model cell that locally expresses biospecific functions.

Serum carbohydrate antigen elevations in endometrial adenocarcinomas: characterization of DU-PAN-2 expression as a tumor marker.

To characterize serum elevations of carbohydrate antigens; DU-PAN-2, CA19-9, sialyl Lewisx and CA125 in endometrial adenocarcinomas (EMACs), particularly focusing on the clarification of DU-PAN-2 expression profiles. Sixty-four resected EMACs of endometrioid type were used. The preoperative serum values of four markers were measured and comparatively analyzed regarding the relationship between histological grade and clinicopathological stage. The overall ratios of positive cases were 26.2% for DU-PAN-2, 25.0% for CA19-9, 13.6% for sialyl Lewisx, and 35.5% for CA125. DU-PAN-2 decreased as the grading went up (G1: 410.3 +/- 243.8 to G3: 246.7 +/- 90.0 U/mL), however, the reverse was true with CA19-9 (G1: 123.9 +/- 147.4 to G3: 320.0 +/- 180.0 U/mL). Sialyl Lewisx showed a strong tendency towards high elevation in G1 (346.3 +/- 102.6 U/mL), compared to G3 (< 2.5 U/mL). CA125 increased markedly as the grading went up (G1: 43.5 +/- 6.3 to G3: 578.0 +/- 10.0 U/mL). During staging-up from I + II to III + IV, the positive ratios inclined in all four markers as follows: DU-PAN-2, 18.4-53.3%; CA19-9, 20.4-40.0%; sialyl Lewisx, 11.4-22.2%; CA125, 31.8-44.4%. Serum elevations and positive ratios were correlated for DU-PAN-2, CA19-9 and CA125, while the reverse relationship was found for sialyl Lewisx. It is suggested that DU-PAN-2 tends to be produced more in well-differentiated components of EMACs than in poorly differentiated ones. Since approximately half the cases with EMAC were serologically positive for DU-PAN-2 in stage III + IV, the marker is believed to be of much use for monitoring the cases with an extrauterine extent.

Mucins and mucin binding proteins in colorectal cancer.

Mucins are high-molecular weight epithelial glycoproteins with a high content of clustered oligosaccharides O-glycosidically linked to tandem repeat peptides rich in threonine, serine, and proline. There are two structurally and functionally distinct classes of mucins: secreted gel-forming mucins (MUC2, MUC5AC, MUC5B, and MUC6) and transmembrane mucins (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC17), although the products of some MUC genes do not fit well into either class (MUC7, MUC8, MUC9, MUC13, MUC15, MUC16). MUC1 mucin, as detected immunologically, is increased in expression in colon cancers, which correlates with a worse prognosis. Expression of MUC2 secreted gel-forming mucin is generally decreased in colorectal adenocarcinoma, but preserved in mucinous carcinomas, a distinct subtype of colon cancer associated with microsatellite instability. Another secreted gel-forming mucin, MUC5AC, a product of normal gastric mucosa, is absent from normal colon, but frequently present in colorectal adenomas and colon cancers. The O-glycosidically linked oligosaccharides of mucins can be described in terms of core type, backbone type, and peripheral structures. Colon cancer mucins have differences in both core carbohydrates and in peripheral carbohydrate structures that are being investigated as diagnostic and prognostic markers, and also as targets for cancer vaccines. Colon cancer mucins typically have increases in three core structures: Tn antigen (GalNAcalphaThr/Ser), TF antigen (Galbeta3GalNAc) and sialyl Tn (NeuAcalpha6GalNAc). The type 3 core (GlcNAcbeta3Ga1NAc) predominant in normal colonic mucin is lacking in colon cancer mucins. There are cancer-associated alterations in the peripheral carbohydrates of colonic mucins including a decrease in O-acetyl-sialic acid and a decrease in sulfation. There are, however, cancer-associated increases in sialyl LeX and related structures on mucins and other glycoproteins that can serve as ligands for selectins, increasing the metastatic capacity of colon cancer cells. The endogenous galactoside-binding protein galectin-3, which is expressed at higher levels in colon cancers than normal colon, binds to colon cancer mucin as well as other glycoproteins. Interference of the binding of selectins and galectin-3 to mucin may show therapeutic or preventative promise for colon cancer.

Distinct Populations of Forebrain Neural Stem and Progenitor Cells Can Be Isolated Using Side-Population Analysis

The absence of stem cell-specific markers has posed challenges to the identification and isolation of stem cells. We report the isolation of a discrete and highly enriched population of neural stem cells from clonally derived colonies of neural stem cell and progenitor cells (neurospheres) after exposure to the fluorescent DNA binding dye Hoeschst 33342 and subsequent analysis via dual wavelength flow cytometry. The low fluorescent side population comprised only 3.6% of all live cells sorted yet contained >99% of all the neural stem cells as assayed by the formation of neurospheres in culture. Most neurosphere-derived cells are progenitor cells, and these are found within the higher fluorescence (non-side population) fraction. The isolation of a highly enriched population of self-renewing, multipotential neural stem cells was seen from both adult- and embryonic-derived neurospheres; however, the relative percentage of cells comprising the side-population and the mechanism of dye efflux varied between adult and embryonic donor tissue. Combining the side-population analysis with markers recently shown to enrich for neural stem cells afforded no further enrichment in the case of peanut agglutinin expression and size criteria; however, when the side-population analysis was combined with Lewis X (LeX) expression, a slight enrichment was seen over side-population analysis alone.