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Objectives: Despite successful antiretroviral (ARV) therapy, people living with HIV (PLWH) may show signs of premature/accentuated aging. We compared established biomarkers of aging in PLWH, appropriately-chosen HIV-negative individuals, and blood donors, and explored factors associated with biological age advancement.Design: Cross-sectional analysis of 134 PLWH on suppressive ARV therapy, 79 lifestyle-comparable HIV-negative controls aged ≥45 years from the Co-morBidity in Relation to AIDS (COBRA) cohort, and 35 age-matched blood donors (BD).Methods: Biological age was estimated using a validated algorithm based on ten biomarkers. Associations between ‘age advancement’ (biological minus chronological age) and HIV status/parameters, lifestyle, cytomegalovirus (CMV), hepatitis B (HBV) and hepatitis C virus (HCV) infections were investigated using linear regression.Results: The average (95% CI) age advancement was greater in both HIV-positive [13.2 (11.6, 14.9) years] and HIV-negative [5.5 (3.8, 7.2) years] COBRA participants compared to BD [-7.0 (-4.1, -9.9) years, both p's < 0.001)], but also in HIV-positive compared to HIV-negative participants (p < 0.001). Chronic HBV, higher anti-CMV IgG titer and CD8+ T-cell count were each associated with increased age advancement, independently of HIV-status/group. Among HIV-positive participants, age advancement was increased by 3.5 (0.1, 6.8) years among those with nadir CD4+ < 200 cells/μL and by 0.1 (0.06, 0.2) years for each additional month of exposure to saquinavir.Conclusions: Both treated PLWH and lifestyle-comparable HIV-negative individuals show signs of age advancement compared to BD, to which persistent CMV, HBV co-infection and CD8+ T-cell activation may have contributed. Age advancement remained greatest in PLWH and was related to prior immunodeficiency and cumulative saquinavir exposure. published

Background The optimum method of defining cognitive impairment in virally suppressed people living with HIV is unknown. We evaluated the relationships between cognitive impairment, including using a novel multivariate method (NMM), patient– reported outcome measures (PROMs), and neuroimaging markers of brain structure across 3 cohorts. Methods Differences in the prevalence of cognitive impairment, PROMs, and neuroimaging data from the COBRA, CHARTER, and POPPY cohorts (total n = 908) were determined between HIV-positive participants with and without cognitive impairment defined using the HIV-associated neurocognitive disorders (HAND), global deficit score (GDS), and NMM criteria. Results The prevalence of cognitive impairment varied by up to 27% between methods used to define impairment (eg, 48% for HAND vs 21% for NMM in the CHARTER study). Associations between objective cognitive impairment and subjective cognitive complaints generally were weak. Physical and mental health summary scores (SF-36) were lowest for NMM-defined impairment (P < .05). There were no differences in brain volumes or cortical thickness between participants with and without cognitive impairment defined using the HAND and GDS measures. In contrast, those identified with cognitive impairment by the NMM had reduced mean cortical thickness in both hemispheres (P < .05), as well as smaller brain volumes (P < .01). The associations with measures of white matter microstructure and brain-predicted age generally were weaker. Conclusion Different methods of defining cognitive impairment identify different people with varying symptomatology and measures of brain injury. Overall, NMM-defined impairment was associated with most neuroimaging abnormalities and poorer self-reported health status. This may be due to the statistical advantage of using a multivariate approach., We have previously described a novel multivariate method (NMM) with theoretical statistical advantages over existing methods, which we assessed here in 3 cohorts of people living with HIV. Overall, NMM-defined impairment was associated with most neuroimaging abnormalities and poorer self-reported health status.

Background Brain structural abnormalities have been reported in persons living with human immunodeficiency virus (HIV; PLWH) who are receiving suppressive combination antiretroviral therapy (cART), but their pathophysiology remains unclear. Methods We investigated factors associated with brain tissue volumes and white matter microstructure (fractional anisotropy) in 134 PLWH receiving suppressive cART and 79 comparable HIV-negative controls, aged ≥45 years, from the Comorbidity in Relation to AIDS cohort, using multimodal neuroimaging and cerebrospinal fluid biomarkers. Results Compared with controls, PLWH had lower gray matter volumes (−13.7 mL; 95% confidence interval, −25.1 to −2.2) and fractional anisotropy (−0.0073; 95% confidence interval, −.012 to −.0024), with the largest differences observed in those with prior clinical AIDS. Hypertension and the soluble CD14 concentration in cerebrospinal fluid were associated with lower fractional anisotropy. These associations were independent of HIV serostatus (Pinteraction = .32 and Pinteraction = .59, respectively) and did not explain the greater abnormalities in brain structure in relation to HIV infection. Conclusions The presence of lower gray matter volumes and more white matter microstructural abnormalities in well-treated PLWH partly reflect a combination of historical effects of AIDS, as well as the more general influence of systemic factors, such as hypertension and ongoing neuroinflammation. Additional mechanisms explaining the accentuation of brain structure abnormalities in treated HIV infection remain to be identified.

The promyelocytic human cell line U937, cultured in the presence of TPA and/or vit. D3, differentiates to monocytes and to macrophage-like cells. A potent stimulus for differentiation is represented also by colony stimulating factor-1 (CSF-1). Since this factor is a strong inducer of PGH synthase in human monocytes, we have investigated whether this event may be connected to the differentiation of U937. We have found that TPA, in the presence of serum, increased the production of thromboxane B2 (TXB2) 4-5 fold, while DMSO, which induced differentiation to neutrophils, was not active. Here we report studies indicating that the effect of protein and RNA synthesis inhibitors on prostanoid production, in cells incubated in the presence of CSF-1 (or FCS), can be correlated with an inductive event carried out by the growth factor, as demonstrated by the use of Western and Northern blotting procedures. However, while in human monocytes PGH-s and its mRNA are absent in controls and are expressed at high levels in CSF-1 stimulated cells, in U937 cells exposed to TPA, PGH-s mRNA was clearly detected by Northern blots, but its translation product was expressed at low level, and cells generated low amounts of TXA2 (13% of maximal production). After incubation with CSF-1 (or FCS) mRNA levels were only slightly modified, but large amounts of TXA2 accumulated in the medium. We have interpreted these findings by suggesting that CSF-1 is capable not only of regulating the expression of the gene encoding PGHs, but also of acing translationally to regulate the expression of its mature mRNA.

Sialyl Lewis x (sLex) is a selectin ligand whose overexpression in epithelial cancers mediates metastasis formation. The molecular basis of sLex biosynthesis in colon cancer tissues is still unclear. The prerequisite for therapeutic approaches aimed at sLex down-regulation in cancer, is the identification of rate-limiting steps in its biosynthesis. We have studied the role of α1,3-fucosyltransferases (Fuc-Ts) potentially involved in sLex biosynthesis in specimens of normal and cancer colon as well as in experimental systems. We found that: (i) in colon cancer, but not in normal mucosa where the antigen was poorly expressed, sLex correlated with a Fuc-T which, like Fuc-TVI, was active on 3′sialyllactosamine at a low concentration (Fuc-T SLN ); (ii) competitive RT-PCR analysis revealed that the level of Fuc-T mRNA expression in both normal and cancer colon was Fuc-TVI > Fuc-TIII > Fuc-TIV; Fuc-TV and Fuc-TVII expression was negligible; (iii) sLex was expressed only by the gastrointestinal cell lines displaying both Fuc-TVI mRNA and Fuc-T SLN activity, but not by those expressing only Fuc-TIII mRNA; (iv) transfection with Fuc-TVI cDNA, but not with Fuc-TIII cDNA, induced sLex expression in gastrointestinal cell lines; (v) Fuc-TVI knock-down with specific siRNA induced down-regulation of Fuc-TVI mRNA and Fuc-T SLN activity and a dramatic inhibition of sLex expression. These data indicate that in colon cancer tissues Fuc-TVI is a key regulator of sLex biosynthesis which can be the target of RNA-interference-based gene knock-down approaches.

Background Glycosylation is increasingly recognized as one of the most relevant postranslational modifications. Sialic acids are negatively charged sugars which frequently terminate the carbohydrate chains of glycoproteins and glycolipids. The addition of sialic acids is mediated by sialyltransferases, a family of glycosyltransferases with a crucial role in cancer progression. Scope of the review To describe the phenotypic and clinical implications of altered expression of sialyltransferases and of their cognate sialylated structures in cancer. To propose a unifying model of the role of sialyltransferases and sialylated structures on cancer progression. Major conclusions We first discuss the biosynthesis and the role played by the major cancer-associated sialylated structures, including Thomsen–Friedenreich-associated antigens, sialyl Lewis antigens, α2,6-sialylated lactosamine, polysialic acid and gangliosides. Then, we show that altered sialyltransferase expression in cancer, consequence of genetic and epigenetic alterations, generates a flow of information toward the membrane through the biosynthesis of aberrantly sialylated molecules (inside-out signaling). In turn, the presence of aberrantly sialylated structures on cell membrane receptors generates a flow of information toward the nucleus, which can exacerbate the neoplastic phenotype (outside-in signaling). We provide examples of self-fueling loops generated by these flows of information. General significance Sialyltransferases have a wide impact on the biology of cancer and can be the target of innovative therapies. Our unified view provides a conceptual framework to understand the impact of altered glycosylation in cancer.

International audience; BACKGROUND:The histo-blood group antigens are carbohydrate structures present in tissues and body fluids, which contribute to the definition of the individual immunophenotype. One of these, the Sd(a) antigen, is expressed on the surface of erythrocytes and in secretions of the vast majority of the Caucasians and other ethnic groups.SCOPE OF REVIEW:We describe the multiple and unsuspected aspects of the biology of the Sd(a) antigen and its biosynthetic enzyme β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) in various physiological and pathological settings.MAJOR CONCLUSIONS:The immunodominant sugar of the Sd(a) antigen is a β1,4-linked N-acetylgalactosamine (GalNAc). Its cognate glycosyltransferase B4GALNT2 displays a restricted pattern of tissue expression, is regulated by unknown mechanisms - including promoter methylation, and encodes at least two different proteins, one of which with an unconventionally long cytoplasmic portion. In different settings, the Sd(a) antigen plays multiple and unsuspected roles. 1) In colon cancer, its dramatic down-regulation plays a potential role in the overexpression of sialyl Lewis antigens, increasing metastasis formation. 2) It is involved in the lytic function of murine cytotoxic T lymphocytes. 3) It prevents the development of muscular dystrophy in various dystrophic murine models, when overexpressed in muscular fibers. 4) It regulates the circulating half-life of the von Willebrand factor (vWf), determining the onset of a bleeding disorder in a murine model.GENERAL SIGNIFICANCE:The expression of the Sd(a) antigen has a wide impact on the physiology and the pathology of different biological systems.

Apoptosis profoundly alters the carbohydrate layer coating the membrane of eukaryotic cells. Previously we showed that apoptotic cells became reactive with the alpha2, 6-sialyl-specific lectin from Sambucus nigra (SNA), regardless of their histological origin and the nature of the apoptotic stimulus. Here we reveal the basis of the phenomenon by showing that in apoptotic cancer cell lines SNA reactivity was mainly associated with a 67 kDa glycoprotein which we identified by MALDI-TOF/TOF and immunoblot analysis as bovine vitronectin (bVN). bVN was neither present in non-apoptotic cells, nor in cells induced to apoptosis in serum-free medium, indicating that its uptake from the cell culture serum occurred only during apoptosis. The bVN molecules associated with apoptotic cancer cell lines represented minor isoforms, lacking the carboxyterminal sequence and paradoxically containing a few alpha2,6-linked sialic acid residues. Despite their poor alpha2,6-sialylation, these bVN molecules were sufficient to turn apoptotic cells to SNA reactivity, which is a late apoptotic event occurring in cells positive to both annexin-V and propidium iodide. Unlike in cancer cell lines, the major bVN form taken up by apoptotic neutrophils and mononuclear cells was a 80 kDa form. In apoptotic SW948 cells we also detected the alpha2,6-sialylated forms of the stress-70 mitochondrial precursor (mortalin) and of tubulin-beta2C. These data indicate that the acquisition of vitronectin isoforms from the environment is a general, although cell specific phenomenon, potentially playing an important role in post-apoptotic events and that the alpha2,6- sialylation of intracellular proteins is a new kind of posttranslational modification associated with apoptosis.

The process of cell transformation induces profound alterations in the glycosylation pattern of the cells. Often, the carbohydrate structures formed by cancer cells resemble those expressed by the corresponding normal tissue during the fetal life (onco-developmental regulation) and are largely tissue-specific. However, some structures appear to be widely expressed by cancers of different histological origin. This is probably due to two main reasons: first, their biosynthesis is strictly controlled by the mechanisms altered in cell transformation (i.e. activation of oncogenes, inactivation of tumour-suppressor genes, altered pattern of epigenetic regulation); second, their expression provide cancer cells with a growth advantage, resulting in the selection of cells expressing a given antigen during tumour growth. In the following sections, we will first describe some of the most widely expressed cancer-related carbohydrate structures (Section 2), then we will discuss how the altered mechanisms controlling cell growth in cancer influence the glycosylation machinery (Section 3). Last, we will discuss how the cancer-related carbohydrate structures modify the cancer cell phenotype (Section 4).

Sialyl Lewis x (sLex) is a well known selectin ligand involved in metastasis. The molecular bases of its overexpression in colon cancer are still unclear. Here we show that FucT-VI is the main fucosyltransferase responsible for sLex biosynthesis in colonic tissues and a major regulator of sLex expression in colon cancer because: 1) in colon cancer specimens and cell lines sLex expression correlates with the activity of a fucosyltransferase able to fucosylate 3’ sialyllactosamine (3’SLN) at a low (0.5 mM) acceptor concentration. 2) In cells transiently transfected with FucT genes only FucT-VI displays this property. 3) In normal and cancer colon, FucT-VI transcript shows the highest level of expression (about 200 fg/pg actin, vs. 30 for FucT-III, 10 for FucT-IV

Many observations have reported glycosylation changes associated with apoptosis in different biological systems, although none of these has shown any general significance. In this work, we show that in cell lines from different histological origin, (colon, breast, pancreas, and bladder cancer) as well as in normal human and mice neutrophils, apoptosis is accompanied by the exposure of sugar chains recognized by the lectin from Sambucus nigra (SNA), specific for Sia alpha 2,6Gal/GalNAc structures. Also, cells undergoing primary necrosis induced by heat treatment (56 degrees C, 30 min) expose specifically binding sites for SNA. While this modification is recognized also by the lectin from the mushroom Polyporus squamosus, which is highly specific for alpha2,6-sialylated lactosamine, no significant changes were detected in the binding of lectins specific for other carbohydrate structures, such as those from Phaseolus vulgaris, Arachis hypogea, and Maackia amurensis. The binding of SNA to apoptotic/necrotic cells is inhibited by neuraminidase treatment and by alpha2,6-sialylated compounds. In apoptotic, but not in necrotic SW948 cells, SNA reactivity is specifically associated with 65, 69, and 87 kDa glycoproteins. The exposure of SNA-reactive chains by apoptotic/necrotic cells occurs also in cells not expressing sialyltransferases ST6Gal.1 or ST6Gal.2 and is largely independent of the presence of alpha2,6-sialylated lactosaminic chains on the surface of preapoptotic cells. In neutrophils from ST6Gal.1 knock-out mice, the apoptosis-related increase in SNA reactivity is reduced but not abolished. These data demonstrate that apoptosis and primary necrosis induce a specific glycosylation change independent of the cell type and nature of the stimulus.

Apoptosis is a tightly regulated process of cell death which results in cell fragmentation and removal of demised cells by phagocytosis. The several glycosylation changes described in apoptotic cells, usually by means of fluorescent lectins, are often cell specific and the identification of apoptosis-associated carbohydrate changes of general significance is still lacking. Sambucus nigra agglutinin (SNA) is a widely used tool for the detection of 2,6-sialylated lactosamine, a carbohydrate epitope often associated with cancer, which is the product of sialyltransferase ST6Gal.1. In this study, we show that cell lines of different histological origin, such as colon, breast, pancreas and bladder cancer undergoing apoptosis display a strong increase of SNA reactivity, through a ST6Gal.1-independent mechanism. This change is specific, in that the reactivity with other lectins, such as Maakia amurensis lectin, peanut agglutinin and leukoagglutinin does not show any consistent change. Surprisingly, also a heat treatment which induces primary necrosis causes a specific increase of SNA reactivity. In the colon cancer cell lines SW48 and SW948 which are basically devoid of measurable ST6Gal.1 activity and which express very low levels of SNA reactivity, apoptosis- or necrosis-inducing treatments induce increased SNA reactivity but do not activate the dormant ST6Gal.1 activity. The spontaneous apoptosis occurring in human neutrophiles at the end of their brief life, also causes a strong increase of SNA reactivity. Together, these data indicate that SNA detects specifically a change occurring in apoptotic and necrotic cells, regardless their histological origin and the nature of the apoptotic stimulus.

Sialyl Lewis X (sLex) is a well known carbohydrate antigen whose overexpression in cancer correlates with metastasis. In colon cancer, the molecular bases of sLex overexpression remain elusive. We have investigated sLex expression in normal and cancer colonic tissues and in cell lines as a function of the expression of fucosyltransferases and beta4GalNAcT-II. This enzyme, which is downregulated in colon cancers, is responsible for the biosynthesis of the Sda antigen [Siaalpha2,3(GalNAcbeta1,4)Galbeta1,4GlcNAc], whose biosynthesis competes with that of sLex. In colon cancers and cell lines, sLex expression correlates with a fucosyltransferase activity able to fucosylate 3'sialyllactosamine at low (0.5 mM) concentration. Transfection experiments with FucT-III, IV, V, VI and VII cDNAs in COS-7 cells indicate that only FucT-VI displays this property. In gastrointestinal cell lines, high levels of this fucosyltransferase activity are shown by cell lines expressing high levels of the FucT-VI transcript. In normal colon, sLex antigen is detectable upon de-acetylation but its expression does not correlate with any fucosyltransferase activity or transcript. Rather, we observed a significant correlation between sLex and the ratio between fucosyltransferase activity with 0.5 mM 3'sialyllactosamine and beta4GalNAcT-II activity. These data suggest that in both normal and cancer colonic tissues the biosynthesis of sLex is mainly due to FucT-VI or to a fucosyltransferase with similar kinetic properties, unknown at present. In normal colon, but not in colon cancers, the fucosyltransferase activity above described is counteracted by competing glycosyltransferases, as the high beta4GalNAcT-II activity that synthesizes the alternative Sda antigen.

The carbohydrate determinants Sd(a) and sialyl Lewis x (sLex) both result from substitution of an alpha2,3-sialylated type 2 chain: the first with an N-acetylgalactosamine (GalNAc) beta1,4-linked to Gal and the second by an alpha1,3-linked fucose on N-acetylglucosamine. The Sd(a) antigen is synthesized by Sd(a) beta1,4-N-acetylgalactosaminyltransferase II (beta4GalNAcT-II), which is downregulated in colon cancer, whereas sLex is a cancer-associated antigen. In view of the possible competition between beta4GalNAcT-II and the fucosyltransferases (FucTs) synthesizing the sLex antigen, we investigated whether beta4GalNAcT-II acts as a negative regulator of sLex expression in colon cancer. beta4GalNAcT-II cDNA, when expressed in LS174T colon cancer cells, induces the expression of the Sd(a) antigen, a dramatic inhibition of sLex expression on cell membranes, and the replacement of sLex with the Sd(a) antigen on 290 kDa glycoproteins. Unexpectedly, in colorectal cancer specimens, beta4GalNAcT-II and sLex show a direct relation. The reasons appear to be (i) Sd(a) and sLex antigens are expressed by different glycoproteins of 340 and 290 kDa, respectively; (ii) the activity of alpha1,3-FucTs on 3'-sialyllactosamine parallels that of beta4GalNAcT-II; and (iii) both beta4GalNAcT-II and FucT activities parallel sLex expression. Quantitative reverse transcription-polymerase chain reaction analysis reveals that the transcripts of beta4GalNAcT-II and those of FucT-III and FucT-VII are positively correlated. These data indicate that in colon cancer tissues, the sLex antigen is regulated mainly by the total FucT activity on 3'-sialyllactosamine acceptors and that beta4GalNAcT-II can inhibit sLex expression in an experimental model, although not in colon cancer tissues.

Beta-Galactoside alpha2,6 sialyltransferase (ST6Gal.I) is the principal sialyltransferase responsible for the biosynthesis of the sialyl alpha2,6-galactosyl linkage. This enzyme and its cognate glycosidic structure are overexpressed in several malignancies and are related to cancer progression. The expression of the enzyme is regulated mainly through the expression of three principal mRNA species differing in the 5' untranslated exons. The form known as YZ is considered associated with the basal expression of the gene, while forms H and X are specific of the liver and of B-lymphocytes, respectively. Using a panel of human cancer cell lines we have studied the expression of ST6Gal.I activity by two different methods, the expression of alpha2,6-sialylated sugar chains by the lectin from Sambucus nigra (SNA) and the expression of the different mRNA species by RT-PCR using oligonucleotide primers complementary to the isoform-specific regions. We report that very high levels of ST6Gal.I activity result in high levels of SNA reactivity and are associated with the expression of the H transcript in colon and liver cell lines and of the X transcript in B cells.

The histo-blood group carbohydrate determinant Sda is expressed by about 95% of the individuals of Caucasian origin. The addition of the immunodominant sugar, a beta1,4-linked N-acetylgalactosamine, is mediated by Sda beta1,4 N-acetylgalactosaminyltransferase (b4GalNAc-T). The human b4GalNAc-T encodes two transcripts diverging in exon 1: one (long form) encodes a polypeptide with an extremely long cytoplasmic domain of 67 aminoacids while the second (short form) predicts a protein with a cytoplasmic domain of 7 aminoacids. The activity of b4GalNAc-T decreases in colorectal cancer at a very variable extent among patients. Sialyl Lewis x (sLex) is a major ligand of selectins and, if ectopically expressed by tumours, can mediate metastatization. We have permanently expressed the two b4GalNAc-T forms in the human colon cancer cell line LS174T and found that the short form induces a higher level of enzyme activity than the long form. Both forms induce expression of the Sda antigen and inhibit the expression of the sLex, very likely because of the competition between b4GalNAc-T and the fucosyltransferases which synthesize the sLex. The two b4GalNAc-T forms, fused with the green fluorescent protein (GFP) localize largely to the Golgi apparatus although a part of the enzyme molecules was present also in non-Golgi compartments. In normal human colon the Sda antigen is mainly expressed by goblet cells and by the well differentiated enterocytes of the apical portion of the gland. When the level of b4GalNAc-T activity was correlated with sLex expression in colon cancer specimens, we observed a direct relationship instead of an inverse relationship, as expected on the basis of the in vitro study. These data indicate that, although b4GalNAc-T has the potential to inhibit sialyl Lewis x expression, it does not play this role in colon carcinogenesis.

Beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I), the enzyme which adds sialic acid in alpha2,6-linkage on lactosaminic termini of glycoproteins, is frequently overexpressed in cancer, but its relationship with malignancy remains unclear. In this study, we have investigated the phenotypic changes induced by the expression of alpha2,6-sialylated lactosaminic chains in the human colon cancer cell line SW948 which was originally devoid of ST6Gal.I. Clones derived from transfection with the ST6Gal.I cDNA were compared with untransfected cells and mock transfectants. The ST6Gal.I-expressing clones show (1) increased adherence to fibronectin and collagen IV but not to hyaluronic acid. Treatment with Clostridium perfrigens neuraminidase reduces the binding to fibronectin and collagen IV of ST6Gal.I-expressing cells but not that of ST6Gal.I-negative cells; (2) accumulation and more focal distribution of beta1 integrins on the cell surface; (3) different distribution of actin fibers; (4) flatter morphology and reduced tendency to multilayer growth; (5) improved ability to heal a scratch wound; (6) reduced ability to grow at the subcutaneous site of injection in nude mice. Our data suggest that the presence of alpha2,6-linked sialic acid on membrane glycoconjugates increases the binding to extracellular matrix components, resulting in a membrane stabilization of beta1 integrins, further strengthening the binding. This mechanism can provide a basis for the flatter morphology and the reduced tendency to multilayer growth, resulting in a more ordered tissue organization. These data indicate that in the cell line SW948, the effect of ST6Gal.I expression is consistent with the attenuation of the neoplastic phenotype.

The biosynthesis of the Sda histo-blood group antigen [Siaalpha2,3(GalNAcbeta1,4)Galbeta1,4GlcNAc-R], is catalyzed by Sda beta1,4GalNAc transferase (beta1,4GalNAc-T). This enzyme, which catalyzes the addition of GalNAc, the immunodominant sugar, to the Gal residue of an alpha2,3-sialylated type 2 chain, is highly expressed in normal colon but it is dramatically downregulated in colon cancer. The acceptor structure of beta1,4GalNAc-T is also a precursor of the biosynthesis of the sialyl Lewis x (sLex) antigen [Siaalpha2,3Galbeta1,4(Fuca1,3)GlcNAc-R], a major ligand for adhesion molecules of the selectin family whose ectopic expression in cancers favours metastasis formation. The fact that the precursor structures of the Sda and the sLex antigens are identical suggests a competition in the biosynthesis of the two structures and that the downregulation of beta1,4GalNAc-T opens the way to the biosynthesis of sLex, favouring the metastatic progression of colon cancer. The two beta1,4GalNAc-T mRNA species cloned to date differ in the first exon and predict two polypeptides with a different cytoplasmic tail: one encodes a polypeptide with an exceptionally long cytoplasmic sequence of 67 aminoacids, the other encodes a polypeptide with a cytoplasmic tail of 7 residues. To investigate the relationship between beta1,4GalNAc-T and sLex expression, we have stably expressed the two forms in the human colorectal cancer cell line LS174T, which expresses good level of sLex. Compared with mock-transfectants, the clones expressing either form of beta1,4GalNAc-T show a dramatic downregulation of sLex expression in FACS analysis, demonstrating that the level of expression of beta1,4GalNAc-T modulates the biosynthesis of sLex, in vitro.

The addition of sialic acid in alpha2,6-linkage to lactosaminic termini of glycoproteins is mainly mediated by beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I). This enzyme and its cognate oligosaccharide structure are frequently overexpressed in cancer and are associated with increased malignancy but the cellular and molecular bases of this relationship are not clear. In this study, we have investigated the role of ST6Gal.I in the in vivo growth. First, we have xenografted in nude mice colon cancer cell lines and analyzed the expression of ST6Gal.I and of alpha2,6-sialylated sugar chains in the xenograft-derived (XD) cell lines. Compared with parental cell lines, the XD cell lines express dramatically increased levels of ST6Gal.I mRNA, detected by RT-PCR analysis, and enzyme activity, as well as an increased reactivity with the alpha2,6-sialyl-specific lectin from Sambucus nigra (SNA) suggesting either a selection of the cells expressing higher levels of ST6Gal.I. Then, we investigated the in vivo growth of clones derived from transfection with the ST6Gal.I cDNA of the colon cancer cell line SW48, which was originally devoid of ST6Gal.I expression. Compared with mock-transfectants, ST6Gal.I-transfectants form more rapidly growing tumours. Moreover, when an identical number of mock- and ST6Gal.I-transfected cells was mixed and injected in the nude mice, the xenografts and the xenograft-derived cell lines were comprised only of SNA-positive ST6Gal.I-transfectants, while mock-transfected cells were usually not present in the xenograft. These results indicate that during the growth of the tumour the cells expressing high levels of alpha2,6-sialylated glycoconjugates are positively selected because of a growth advantage.

beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I), the enzyme which adds sialic acid in alpha2,6-linkage on lactosaminic termini of glycoproteins, is frequently overexpressed in cancer. This change is associated with increased malignancy but the molecular bases of this relationship remain elusive. In this study, we have investigated the phenotypic changes related with overexpression of alpha2,6-sialylated lactosaminic chains by using clones derived from transfection of the human colon cancer cell line SW948 with the ST6Gal.I cDNA or mock transfected. Compared with untransfected cells and mock-transfectants, the ST6Gal.I-expressing clones show increased adherence to fibronectin and collagen IV but not to hyaluronic acid. Neuraminidase treatment resulted in reduced binding to fibronectin and collagen IV of a ST6Gal.I-expressing clone but in no effect on a mock-transfectant. While untransfected and mock-transfected cells tend to form multistratified tissues, ST6Gal.I-expressing clones show a flatter morphology and the tendency to grow as a monolayer. FACS analysis revealed that all ST6Gal.I-expressing clones display higher amounts of beta1 integrins on the cell surface; this difference is not supported by differences at the beta1 integrin mRNA level and is lost when cells are left in suspension for several hours, suggesting a mechanism of membrane stabilization of beta1 integrins dependent on the presence of alpha2,6-linked sialic acid. Our data support a model in which the presence of alpha2,6-linked sialic acid on membrane glycoconjugates increases the binding to extracellular matrix components, resulting in membrane stabilization of beta1 integrins, further strengthening the binding. This mechanism provides a basis for the altered phenotype associated with ST6Gal.I overexpression.

Cell membrane glycoconjugates undergo characteristic changes as a consequence of neoplastic transformation. The cancer-associated carbohydrate structures play key roles in cancer progression by altering the cell-cell and cell-environment interactions. In this review, we will discuss some of the most relevant cancer-associated carbohydrate structures, including the β1,6-branching of N-linked chains, the sialyl Lewis antigens, the α2,6-sialylated lactosamine, the Thomsen-Friedenreich-related antigens and gangliosides. We will describe the mechanisms leading to the expression of these structures and their interactions with sugar binding molecules, such as selectins and galectins. Finally, we will discuss how the glycosylation machinery of the cell is controlled by signal transduction pathways, epigenetic mechanisms and responds to hypoxia.