Biosynthesis of the cancer-related sialyl-alpha 2,6-lactosaminyl epitope in colon cancer cell lines expressing beta-galactoside alpha 2,6-sialyltransferase under a constitutive promoter.

An elevation of beta-galactoside alpha 2,6-sialyltransferase (ST6Gal.I) enzyme activity and an increased alpha 2,6-sialylation of cell membranes are among the most prominent glycosylation changes associated with colon cancer; both modifications correlate with a worse prognosis. In our previous studies, we have frequently observed a discrepancy between the ST6Gal.I level within a colon cancer sample or cell line and the respective level of reactivity with the alpha 2,6-sialyl-specific lectin from Sambucus nigra (SNA). In this study, we have investigated quantitatively the biosynthesis of the sialyl-alpha 2,6-lactosaminyl epitope in two colon cancer cell types expressing the ST6Gal.I cDNA under the control of a constitutive promoter. By measuring the amount of ST6Gal.I mRNA using competitive RT-PCR, the expression of alpha 2,6-sialylated lactosaminic structures with SNA and anti-CDw75 Ig, and the presence of unsubstituted lactosaminic termini by Erythrina cristagalli lectin, we reached the following conclusions: (a) a high proportion of the cell surface lactosaminic termini remains unsubstituted, even in the presence of a very high ST6Gal.I activity. This proportion is strongly dependent on the cell type; (b) ST6Gal.I-transfected colon cancer cells do not express the CDw75 epitope; (c) the level of ST6Gal.I enzyme activity only partially correlates with the mRNA level; (d) despite the control by a constitutive promoter, the ST6Gal.I mRNA is not constantly expressed over time; and (e) a very large portion of the enzyme molecules is secreted in the extracellular milieu. These results indicate that post-transcriptional and post-translational mechanisms play a pivotal role in the control of alpha 2,6-sialylation in colon cancer cells.

Sialyltransferases in cancer.

It has long been known that cancer cells often express more heavily sialylated glycans on their surface and that this feature sometimes correlates with invasion. It is now well established that specific sialylated structures, such as the Thomsen-Friedenreich-related antigens, the sialyl Lewis antigens, the sialyl alpha2-6 lactosaminyl structure, the polysialic acid or some gangliosides, can mediate cellular interactions and are altered in cancer cells. This review summarizes the current knowledge on the cancer-associated alterations in sialyltransferase expression which are often at the basis of the deranged expression of sialylated structures.

Beta-galactoside alpha2,6 sialyltransferase in human colon cancer: contribution of multiple transcripts to regulation of enzyme activity and reactivity with Sambucus nigra agglutinin.

Colon cancer tissues display an increased activity of beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I) and an increased reactivity with the lectin from Sambucus nigra (SNA), specific for alpha2,6-sialyl-linkages. Experimental and clinical studies indicate a contribution of these alterations to tumor progression, but their molecular bases are largely unknown. In many tissues, ST6Gal.I is transcriptionally regulated through the usage of different promoters that originate mRNAs diverging in the 5;-untranslated regions. RT-PCR analysis of 14 carcinoma samples, all expressing an increased ST6Gal.I enzyme activity, and of the corresponding normal mucosa revealed the presence of at least 2 transcripts. One, containing the 5;-untranslated exons, Y+Z, is thought to represent the "housekeeping" expression, and another previously described in hepatic tissues. Both the Y+Z and the hepatic transcripts were detectable in normal and cancer tissues but that latter form had a marked tendency to accumulate in cancer. The extent of alpha2,6-sialylation of glycoconjugates, as determined by SNA-dot blot analysis, was markedly enhanced in all cancer specimens, but the level of reactivity only partially correlated with the level of enzyme expression. Western blot analysis revealed a strikingly heterogeneous pattern of SNA reactivity among cancer tissues. These data indicate that: i) during neoplastic transformation of colonic cells, ST6Gal.I expression may be modulated through a differential promoter usage; ii) the extent of alpha2,6-sialylation of cancer cell membranes is not a direct function of the ST6Gal.I activity, strongly suggesting the existence of other, more complex mechanisms of regulation.

Differential expression of the hepatic transcript of beta-galactoside alpha2,6-sialyltransferase in human colon cancer cell lines.

The activity of beta-galactoside alpha2,6-sialyltransferase (ST6Gal.1), the enzyme responsible for the addition of sialic acid in alpha2,6-linkage to N-acetyllactosaminic (Gal beta1,4GlcNAc) units of glycoconjugates, is increased in the vast majority of colon cancer specimens, and a positive correlation with an invasive phenotype has been suggested by several studies. In many tissues, ST6Gal.1 is regulated mainly at the transcriptional level through the use of different cell-specific promoters which generate transcripts differing in their 5'-untranslated regions. With the aim of understanding the molecular bases of the increased ST6Gal.1 expression in colon cancer, we investigated the expression of mRNA species in colon cancer cell lines and the relationship with enzyme activity and extent of alpha2,6-sialylation of cell glycoproteins. All cell lines examined express the form containing the 5'-untranslated exons Y and Z, typical of the "basal" expression of the gene, while others express also the liver transcript. This indicates that colon cancer cell lines can be grouped according to expression of the liver transcript of ST6Gal.1. The cell lines expressing only the Y+Z form display, in general, a lower activity:mRNA ratio, which might indicate reduced translational efficiency. The level of alpha2,6-sialylation of cell glycoproteins, as determined by reactivity with the Sambucus nigra lectin, is closely associated with the level of enzyme activity.

Expression of beta-galactoside alpha 2,6-sialyltransferase does not alter the susceptibility of human colon cancer cells to NK-mediated cell lysis.

The extent of processing of N-linked oligosaccharides and the sialylation of the target cell membranes has been positively correlated with resistance to lysis mediated by NK cells, but a conclusive evidence has never been reached. Colon cancer tissues express an increased activity of beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1, alpha 2,6ST), which catalyzed the addition of sialic acid in alpha 2,6-linkage to Gal beta 1,4GlcNAc (N-acetyllactosamine) sequences of glycoprotein N-linked chains. The resulting increased level of membrane alpha 2,6-sialylation appears to be related with a more invasive behavior of cancer cells. This phenomenon may depend on a decreased sensitivity of colon cancer cells to NK cells. To obtain conclusive evidence on the role played by sialylation of N-linked chains in determining the target cell susceptibility to NK-mediated lysis, human colon cancer cell lines not expressing sialyltransferases acting on N-linked chains were transfected with a rat alpha 2,6ST cDNA. Stable transfectants expressed different levels of alpha 2,6ST activity, were reactive with the Sambucus nigra lectin, specific for alpha 2,6-linked sialic acid, and compared with control transfectants, showed a remarkable decrease in the number of unsubstituted Gal beta 1,4GlcNAc terminal sequences. The NK susceptibility of these clones was found to be identical to that of control transfectants, either when unstimulated- or IL-2-stimulated lymphocytes were used as effectors. Neuraminidase treatment of target cells does not result in significant changes to NK susceptibility. Our data demonstrate that sialic acid alpha 2,6-linked to N-linked chains of target cell glycoproteins does not play a major role in recognition of the target by human NK cells.

Human colon cancer cell lines permanently expressing alpha 2,6-sialylated sugar chains by transfection with rat beta-galactoside alpha 2,6 sialyltransferase cDNA.

A role of alpha 2,6-linked sialic acid in the development of an invasive phenotype in colon cancer has been suggested by several observations but never conclusively demonstrated. An experimental model to clarify this tissue was established by the creation and characterization of a bank of cell lines that differ mainly, if not exclusively, in the degree of alpha 2,6-sialylation. Human colon cancer cell lines SW48 and SW948, normally unable to elaborate the alpha 2,6-sialyl linkage, were transfected with the beta-galactoside alpha 2,6-sialyltransferase (alpha 2,6ST) cDNA driven by the cytomegaloviral promoter and screened for cell surface alpha 2,6-sialylated sugar chains using fluorescein isothiocyanate-conjugated Sambucus nigra agglutinin (SNA-FITC). A panel of SNA-FITC-positive clones was established that expresses alpha 2,6ST activity of varying degrees. Only the SNA-FITC-positive recombinants express the 1.2 Kb mRNA predicted to be generated from the transfected sequence. No 4.3-4.7 Kb transcripts that are indicative of transcription from the native alpha 2,6ST gene were detected.

Possible involvement of prostaglandin H synthase-1 induction in the differentiation of U-937 cells.

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.

Cell adhesion molecules CD11a and CD18 in blood monocytes in old age and the consequences for immunological dysfunction. Preliminary results.

Adhesion molecules, such as leukocyte-function-associated antigen (LFA-1 or CD11a/CD18), intercellular adhesion molecule 1 (ICAM-1 or CD54) and Hermes antigen (HCAM or CD44), have important roles in many adhesive interactions involving cells of the immune system. Since it has been shown that many immunological alterations were present in aged subjects, we studied the expression and density of these molecules on peripheral blood lymphocytes and monocytes from healthy old subjects. A decrease in monocyte subpopulations bearing CD11a/CD18 and an increase in CD11a/CD18 and and CD44 antigen density on lymphocytes and on monocytes, respectively, were observed. These changes might be an event in the mechanism leading to the decreased lymphocyte proliferative response in vitro and to other immunological dysfunctions reported in old subjects.

Influence of age and health on immune functions and trace elements.

Apparently healthy elderly donors were screened according to a simple protocol that included clinical examination and the determination of hematological and biochemical values. This screening was performed to detect subclinical alterations which might interfere with immune responses and trace element status. The elderly were divided into two groups. The first group consisted of 22 (age 76 +/- 1 years) positively selected elderly (PSE), i.e. healthy subjects with no hematological and laboratory alterations, the second one comprised 13 (age 75 +/- 1 years) negatively selected elderly (NSE). Data were then compared with those obtained from 40 (age 35 +/- 2 years) healthy young controls. In both groups of elderly donors, plasma zinc levels were normal, while plasma copper concentrations were increased. Intracellular values of zinc and copper in mono- and polymorphonuclear cells from both groups of elderly were within reference limits. After in vitro activation, granulocyte chemiluminescence activity was impaired only in NSE. A decrement in the number of circulating CD3 lymphocytes and an increase in CD8d, CD57 cells were found in PSE, while NSE showed an increased number of CD3,DR cells and CD8d, CD57, CD8b,CD57 and CD16,CD56 positive cells. Our results indicate that only plasma copper levels were affected by age, whereas subclinical alterations in hematological or biochemical values appear to impair immune responses in the elderly.

Oral zinc supplementation in Down's syndrome subjects decreased infections and normalized some humoral and cellular immune parameters.

The effect of 4 months of oral zinc supplementation on immune functions in non-institutionalized young female and male Down's syndrome (DS) subjects was studied. Along with plasma levels of zinc, the immune parameters, measured before and after zinc treatment, were plasma levels of thymulin, the percentage and the absolute number of circulating white blood cells, total lymphocytes, lymphocyte subpopulations, the mitogen-induced lymphocyte proliferation, the production of interleukin-2, and the activity of stimulated granulocytes. Some immune parameters were significantly influenced by zinc treatment. In particular, a normalization of thymulin and zinc plasma levels were found in these subjects after zinc supplementation. At the end of the clinical trial, in vitro lymphocyte proliferation and polymorphonuclear activity also increased and reached normal values. Zinc administration exerted a positive clinical effect in these children, since a reduced incidence of infections was found.

Plasmic levels of trace elements and immune functions in the healthy elderly.

Healthy elderly persons were selected according to an admission protocol which included clinical, hematological and biochemical parameters. Plasmic levels of zinc in these subjects were in the normal range, while plasmic copper was higher than that of young controls. The number of circulating lymphocytes and CD3+ cells was decreased; however, the absolute number of CD4+, CD8b, CD8d, CD20+ and CD57+ cells did not differ from that of controls. A decreased lymphocyte response to PHA in serum-free medium cultures was also observed in the healthy elderly persons.

Enhanced DNA repair in lymphocytes of Down syndrome patients: the influence of zinc nutritional supplementation.

Oral zinc supplementation is able to correct zinc deficiency and some immune defects present in Down's syndrome (DS), while other beneficial effects can be predicted because of the broad spectrum of biochemical pathways and the great variety of enzymes which depend on zinc bio-availability. To test if the maintenance of DNA integrity is also affected by zinc supplementation, DNA damage and repair after gamma-radiation was studied by alkaline elution assay in phytohemagglutinin-stimulated lymphocytes from Down's syndrome children before and after an oral zinc supplementation given for 4 months to correct their immune defects. In comparison with lymphocytes from normal children the DNA damage induction after ionizing radiation in DS lymphocytes both before and after zinc supplementation was normal. On the other hand, the rate of DNA repair in DS was highly and significantly accelerated before zinc treatment. After supplementation with zinc sulfate, the DNA repair rate was consistently slowed down becoming similar to that of control subjects. This is the first demonstration that a nutritional intervention in humans is apparently able to modify the biochemical steps which control the rate of DNA repair.

Functional assessment of cellular non-specific and specific immunity in selected healthy elderly.

Thirteen healthy elderly were selected according to a simplified SENIEUR admission protocol including clinical, hematological and biochemical parameters. The goal of this protocol was to limit the influence of diseases and/or medications on the assessment of immune functions in the elderly. Plasma zinc levels of healthy elderly were comparable to those of young subjects. Cellular nonspecific immunity was determined by measuring chemiluminescence (CL) of peripheral blood granulocytes activated by opsonized zymosan particles. CL of granulocytes from healthy elderly was delayed in comparison to that of young controls when autologous serum was used. Lymphocyte proliferation induced by phytohemagglutinin-P (PHA-P) or zinc chloride (ZnCl(2)) in a serum free medium was lower in the elderly than in young controls. Preincubation of lymphocytes with ZnCl(2) before PHA-P stimulation did not restore the impaired proliferative activity of cells from old donors.

Cytotoxicity of, and DNA damage by, active oxygen species produced by xanthine oxidase.

Toxicity to Raji cells of the xanthine oxidase/hypoxanthine system is related to the formation of single-strand DNA breaks. DNA damage was proportional to the concentration of xanthine oxidase and to the time of exposure. It was prevented by the absence of hypoxanthine, or by the presence of allopurinol, or both superoxide dismutase and catalase. The release of 51Cr from damaged cells was detectable 12 h after the inhibition of cloning efficiency and the production of DNA breakage. These data suggest that DNA damage induced by the oxygen products precedes the severe lesion to the cellular membrane.

Immunosuppressive activity of Tamm-Horsfall glycoprotein oligosaccharides: effect of removal of outer sugars and conjugation with a protein carrier.

Tamm-Horsfall (TH) glycoprotein, the major protein of human urine, is, in vitro, a powerful immunosuppressive agent and the activity resides in its oligosaccharide chains. In this study we investigated structural features required for the inhibitory activity of TH glycoprotein oligosaccharides in the one-way mixed lymphocyte reaction (MLR). We found that both high-mannose and complex-type TH glycopeptides, fractionated from Pronase-digested TH glycoprotein, behaved as inhibitors. Sequential exoglycosidase digestion of complex-type TH glycopeptide results in a slight increase of the inhibitory activity, with a maximum after desialylation and beta-galactosidase treatment. These results suggest that the immunosuppressive activity resides in the central portion of TH glycoprotein N-linked oligosaccharides. The conjugation of complex-type TH glycopeptides to a protein carrier, such as bovine serum albumin, greatly enhanced the inhibitory activity. This effect occurred if the TH-glycopeptide conjugate was added to MLR within the first 24 hr. These results indicate that (i) the immunosuppressive activity is strongly dependent on a multivalent interaction between TH oligosaccharides and ligand(s) at the lymphocyte surface; (ii) an early step of cell-cell recognition is the target of the immunosuppressive conjugate; (iii) TH oligosaccharides compete with a carbohydrate recognition system between effector and stimulator cells which contributes to the MLR-induced blastogenesis.

Interleukins and interferons: yin-yang modulators of PGH synthase in human macrophages.

Prostaglandin H synthase (PGHs) not only is an unstable enzyme, mainly when it is challenged with substrate, but its mRNA is one of the shortest lived species so far identified in mammalian cells. Therefore, signals regulating its level are critical for the role it plays in many cells. The expression of genes coding for PGHs appears to be under the control of polypeptide growth factors. This is in accordance with our previous data showing that a colony stimulating factor-1 (CSF-1)-like factor induces the de novo synthesis of PGHs in human monocytes, as demonstrated by immunoblotting. Here we extend this concept by showing that interleukin (IL)-1 alpha behaves as a potent inducer of PGHs in human macrophages, as indicated by the block in its action due to the addition of RNA and protein synthesis inhibitors. Interferons (IFNs) alpha and beta, however, inhibit prostanoid production in a dose-dependent fashion mainly when macrophages activated by serum are tested. Thus, the PGHs system appears to be under a fine control, CSF-1 being the main regulator during the differentiation from pro-monocyte to monocyte and from monocyte to macrophage, and IL-1 (and perhaps IL-2) as well as IFN alpha and beta, the regulators during differentiation and/or proliferation of human macrophages.

Prostanoids as second messengers of polypeptide growth factors.

Prostaglandin H synthase (PGHs), also known as cyclooxygenase, is an unstable enzyme whose mRNA has an half life of 10 minutes. Some polypeptide factors have been reported to induce the enzyme in target cells. We have purified and characterized a component of animal sera which behaves as a potent inducer of human monocyte PGHs. This factor, called serum monocytotropic factor, has been identified in human platelets and it appears to be structurally and biochemically different from identified platelet factors, such as platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), while showing strong similarities to colony stimulating factor 1 (CSF-1), so far undetected in platelets. Moreover, we have shown, by immunoblot analysis, that CSF-1 behaves as a potent and specific inducer of monocyte PGHs. The hypothesis that prostanoids may be considered as second messengers of platelet CSF-1 like factor, as well as of other growth factors and that PGHs induction plays a pivotal role in this process, will be illustrated.

Derangement of non-specific immunity in Down syndrome subjects: low leukocyte chemiluminescence activity after phagocytic activation.

Metabolic activation of peripheral blood leukocytes (chemiluminescence) from 27 children with Down syndrome (DS) and 23 age and sex-matched control children after phagocytic stimulation by opsonized zymosan particles was investigated through a chemiluminescence assay. Using autologous plasma or serum as opsonizing media, phagocytic activity of circulating leukocytes was significantly decreased in DS subjects. A further decrease of phagocytic activity was found in neutrophils from DS children, when normal heterologous plasma or sera were used. On the other hand, sera or plasma from DS subjects significantly increased phagocytic activation of leukocytes from normal donors. In DS subjects opsonizing agents such as serum immunoglobulins and complement fractions were in the normal ranges of concentration. Thus, the impaired chemiluminescence of neutrophils was mainly due to a metabolic impairment at the cellular level. A decreased production of radicals derived from the oxygen metabolism in neutrophils may be an important step of immune derangement leading to the increased incidence of infectious diseases frequently associated with DS.

Immune dysfunction in primary biliary cirrhosis. II. Increased production of prostaglandin E.

In a previous study we observed that after in vitro treatment with indomethacin, lymphocyte response to phytohaemagglutinin (PHA) in primary biliary cirrhosis (PBC) patients was higher than that of controls. We know that indomethacin also inhibits prostanoid production, and thus in the present work we directly measured prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) production by mononuclear cells and monocytes from 12 PBC patients, 11 control subjects, and three control disease patients (alcoholic cirrhosis, AC). PHA-stimulated enriched monocytes from PBC patients produced approximately threefold more PGE2 (after 48 h of culture) than did normal and AC monocytes (P less than 0.05). TXB2 production was similar in all groups studied. We also made cultures in which PBC-purified lymphocytes proliferated better than PBC mononuclear cells (i.e. lymphocytes plus monocytes). Thus, a monocyte population producing PGE2 could be responsible, at least in part, for the hyporesponsiveness to PHA observed in PBC patients.