Carcinoma-related alterations of glycosyltransferases in human tissues.

The glycosyltransferases responsible for catalyzing additions of A, B, and H sugars to cellular acceptors were studied in 23 cases of primary carcinoma. The carcinomas were derived from mouth, tongue, larynx, lung, cervix, esophagus, stomach, and colon. Comparisons of A, B, and H enzymes were made between mucosal extracts from tumor and from normal adjacent tissue and, in the case of gastrointestinal tract, extracts derived from mucosae of individuals free of disease. The most prevalent finding was that of alpha-2-fucosyltransferase (H enzyme) deficiency in tumor extracts from Group A, B, and O patients in relation to the normal tissue counterpart (20 cases). Exceptions were observed in one case of carcinoma of the stomach and in two of seven cases of carcinoma of rectum or sigmoid. In four of nine Group A patients (carcinoma of the mouth, tongue, ascending and transverse colon), N-acetylgalactosaminyltransferases (A enzymes) were demonstrated but were deficient in relation to the normal adjacent counterpart. A enzymes were not demonstrable in normal and tumor extracts from distal colon in five cases. Differences between tumor extracts and normal adjacent tissue were noted in D-galactosyltransferase (B enzyme) derived from carcinomas of larynx and esophagus, but B enzyme was not demonstrated in tumor or normal tissue derived from the sigmoid colon. Study of the normal distribution of H enzyme in gastrointestinal mucosa indicated the presence of relatively high enzyme levels in stomach and upper intestine but low levels in distal colon.

ABH and Lewis blood group expression in colorectal carcinoma.

The expression of ABO(H) and Lewis blood group antigens on 68 colorectal carcinomas from 63 patients was studied by immunohistochemical staining of tissue specimens. The pattern of antigen expression was as follows: ABH was expressed in normal tissue only in secretors and was expressed in the proximal but not distal colon. In tumors, there was net loss of ABH expression in the proximal and net gain in the distal colon. Some nonsecretor tumor tissue expressed ABH. Lewis expression was similar to but less strong than ABH. Its expression occurred only in secretors and in normal epithelium only in the proximal colon. In tumors there was net loss of antigen expression proximally and net gain in the distal colon. There was no expression of Lewisb in tumors of nonsecretors. Lewisa antigen was expressed throughout the normal colon in secretors and nonsecretors with no discernible difference between proximal and distal colon. In tumors, net loss of expression of Lewisa occurred throughout the colon. No inappropriate blood group expression was observed in this study. With few exceptions, H expression paralleled expression of A and B in non-0 patients. Metastatic tumor antigen expression was similar to that of the primary in most cases. Alterations in antigen expression were not clinically or histologically distinctive.

Immunological heterogeneity of carcinoembryonic antigen: immunohistochemical detection of carcinoembryonic antigen determinants in colonic tumors with monoclonal antibodies.

The immunoperoxidase localization of carcinoembryonic antigen (CEA) determinants was studied in colonic adenocarcinomas using four murine monoclonal antibodies to CEA in a bridged avidin:biotin technique. One of the monoclonal antibodies, NP-1, recognizes a CEA epitope shared with the nonspecific cross-reacting antigen and meconium antigen. Two others, NP-2 and NP-3, discriminate two separate CEA epitopes shared with meconium antigen only, whereas NP-4 reacts with a unique determinant expressed on a subpopulation of CEA molecules. The monoclonal antibodies and polyclonal goat antisera against CEA and nonspecific cross-reacting antigen stained columnar epithelial cells in morphologically normal mucosa. Neutrophils were stained by only the NP-1 monoclonal antibody and goat anti-nonspecific cross-reacting antigen antiserum. All moderately differentiated colorectal adenocarcinomas and most of their nodal and liver metastases reacted with the goat antisera and cross-reactive monoclonal antibodies. Thirty % of these primary tumors and most of the nodal and/or liver metastases from six patients with NP-4-positive primary tumors failed to stain with NP-4. These results suggest heterogeneity in the expression of a CEA variant and/or determinant recognized by the NP-4 monoclonal antibody that perhaps identifies a subgroup of colonic cancers which differ in their functional differentiation.

Leukemia-induced alterations of serum glycosyltransferase enzymes.

Studies on blood group A and H glycosyltransferase enzymes in 54 patients with acute myeloid leukemia were carried out on serum derived from blood samples taken prior to treatment, and in 16 cases, further tests were performed during clinical remission and at the time of relapse. The enzyme assay procedures, using low-molecular-weight compounds as sugar acceptors and radioactive nucleotide sugars as the donor substrates, have been described by Chester et al. (Eur. J. Biochem., 69:583, 1976). Abnormally low values of H enzyme (expressed as percentage of radioactive sugar incorporated into product; (that is, 1 to 3%) were observed in practically all presentation sera, but the values reverted to normal levels (3 to 15%) at the time of clinical remission and then became low once more with the development of drug resistance and clinical relapse. A enzyme levels measured in presentation sera which had demonstrated abnormal H enzyme were mostly within the normal range. In 2 of 5 A1 patients; sera and in all of three A2 patients increases in enzyme levels were observed in remission as compared with presentation serum samples. The depression of biosynthetic enzymes in acute leukemic sera could not be accounted for on the basis of competitive inhibitors or catabolic enzymes. It is proposed that changes of serum glycosyltransferase enzymes reflect alterations in a leukemic cell population and that knowledge of these changes may be of value in prognosis in acute leukemia.

Biosynthesis of O-glycans in leukocytes from normal donors and from patients with leukemia: increase in O-glycan core 2 UDP-GlcNAc:Gal beta 3 GalNAc alpha-R (GlcNAc to GalNAc) beta(1-6)-N-acetylglucosaminyltransferase in leukemic cells.

We have studied the biosynthesis of altered O-glycan structures on leukocytes from patients with chronic myelogenous leukemia and with acute myeloblastic leukemia (AML). It has been shown previously that the activity of CMP-NeuAc:Gal beta 1-3GalNAc alpha-R (sialic acid to galactose) alpha(2-3)-sialytransferase (EC 2.4.99.4) is increased in leukocytes from patients with chronic myelogenous leukemia (M. A. Baker, A. Kanani, I. Brockhausen, H. Schachter, A. Hindenburg, and R. N. Taub, Cancer Res., 47: 2763-2766, 1987) and with AML (A. Kanani, D. R. Sutherland, E. Fibach, K. L. Matta, A. Hindenburg, I. Brockhausen, W. Kuhns, R. N. Taub, D. van den Eijnden and M. A. Baker, Cancer Res., 50: 5003-5007, 1990). This increased activity may in part be responsible for the hypersialylation observed in leukemic leukocytes; however, hypersialylation may also be due to changes in underlying O-glycan structures. To test this hypothesis, we have assayed in normal human granulocytes and leukemic leukocytes several glycosyltransferases involved in the synthesis and elongation of the four common O-glycan cores. UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta(1-6)-GlcNAc transferase (EC 2.4.1.102), which synthesizes O-glycan core 2 (GlcNAc beta 1-6[Gal beta 1-3]GalNAc alpha), is significantly elevated in chronic myelogenous leukemia (4-fold) and AML (18-fold) leukocytes relative to normal human granulocytes. Neither normal nor leukemic cells show detectable activities of GlcNAc transferases which synthesize O-glycan core 3 (GlcNAc beta 1-3GalNAc-R) and core 4 (GlcNAc beta 1-6[GlcNAc beta 1-3] GalNAc-R) or the blood group I structure. The beta 3-GlcNAc transferase which elongates core 1 and core 2 was found at low levels in normal granulocytes but was not detectable in leukemic cells. The beta 3-GlcNAc transferase and beta 4-Gal transferase involved in poly-N-acetyllactosamine synthesis, as well as the beta 3-Gal transferase synthesizing core 1 (Gal beta 3 GalNAc), were present in all samples but were significantly increased in patients with AML. The observed changes are consistent with hypersialylation in leukemia.

Human leukemic myeloblasts and myeloblastoid cells contain the enzyme cytidine 5'-monophosphate-N-acetylneuraminic acid:Gal beta 1-3GalNAc alpha (2-3)-sialyltransferase.

We have examined the role of CMP-NeuAc:Gal beta 1-3GalNAc-R alpha(2-3)-sialyltransferase in fresh leukemia cells and leukemia-derived cell lines. Enzyme activity in normal granulocytes using Gal beta 1-3GalNAc alpha-o-nitrophenyl as substrate was 1.5 +/- 0.7 nmol/mg/h whereas activity in morphologically mature granulocytes from 6 patients with chronic myelogenous leukemia (CML) was 4.2 +/- 1.6 nmol/mg/h (P less than 0.05). Myeloblasts from 5 patients with CML in blast crisis showed enzyme activity levels of 6.5 +/- 2.5 nmol/mg/h. From 2 patients with CML, both blasts and granulocytes were obtained, with higher enzyme activity in the patients' blasts (7.1 nmol/mg/h) than in their granulocytes (4.9 nmol/mg/h) in both cases, suggesting that the increase in enzyme activity is related to the differentiation or proliferation status of the CML cells. However, similarly high enzyme levels were also seen in myeloblasts from acute myeloblastic leukemia patients (5.6 +/- 1.4 nmol/mg/h) and in some acute myeloblastic leukemia-derived cell lines (KG1a and HL60), suggesting that increased levels of this enzyme are not directly correlated with the presence of the Ph1 chromosome. This alpha(2-3)-sialyltransferase activity can also be detected in normal peripheral blood lymphocytes and exhibits increased activity in chronic lymphocytic leukemia cells and acute lymphoblastic leukemia. These data suggest that the level of enzyme activity may vary with growth rate and maturation status in myeloid and lymphoid hemopoietic cells. Finally, we have identified a glycoprotein in acute myeloblastic leukemia cells that serves as a substrate for the alpha(2-3)-sialyltransferase. The desialylated form of the glycoprotein was resialylated in vitro by the purified placental form of this alpha(2-3)-sialyltransferase and exhibits a molecular weight of about 150,000.

Fluorescent antibody localization of Microciona prolifera aggregation factor and its baseplate component.

Specific rabbit antisera were prepared against purified aggregation factor and its membrane-associated receptor, baseplate, derived from the marine sponge. Microciona prolifera. They were utilized in conjunction with fluorescent-labeled goat anti-rabbit IgG in an assay to demonstrate the surface localizations of both components. The specificity of antibody preparations for AF and BP was demonstrated through inhibition of the rotation-mediated assay by homotypic antibody. This study confirms the presence of aggregation factor on the surface of disaggregated sponge cells maintained in the presence of the divalent cations, Ca++ and Mg++, and its absence when cells are maintained in Ca++ and Mg++-free seawater. The location of BP could also be demonstrated on the cell surface. Aggregation factors and baseplate appear to be heavily distributed on archeocytes and choanocytes, but are localized less intensely on gray cells. Gray cells are typified by yellowish autofluorescence of their intracellular granules in stained and control preparations. The reaction of anti-Microciona aggregation factor with its homotypic factor appeared to be species specificity judged by immunofluorescence assays and by inhibition of rotation-mediated assay by anti-homotypic AF since antibodies prepared against heterotypic AF preparations were unreactive.

A sulfated proteoglycan aggregation factor mediates amyloid-beta peptide fibril formation and neurotoxicity.

Proteoglycans are associated with senile plaques in Alzheimer's disease and may be involved in A beta fibril formation and plaque formation. In vitro, glycosaminoglycans have been shown to inhibit the proteolysis of A beta fibrils, accelerate formation and maintain their stability. To model their interaction, we investigated the binding of a sulfated proteoglycan derived from a natural source; marine sponge Microciona prolifera aggregation factor (MAF). This species-specific re-aggregation of sponge cells has two functional properties, a Ca2+ independent cell binding activity and a Ca2+ dependent self-aggregation. It has been shown that a novel sulfated disaccharide and a pyruvylated trisaccharide are important in the Ca(2+)-dependent MAF aggregation. Aggregation demonstrated by homophilic binding of MAF subunits may be chemically distinct from other heterotypic binding effects. We investigated A beta-MAF interactions and show that MAF induces a structural transition in A beta 40 and A beta 42 from random to beta-structure as detected by circular dichroism spectroscopy. Electron microscopy revealed that the structural transition correlated with an increase in the number of A beta 40 and A beta 42 aggregated that have a truncated fibrillar morphology. Finally, MAF increased A beta-induced toxicity of nerve growth factor (NGF)-differentiated PC-12 cells in the absence of Ca2+. The addition of Ca2+ to MAF-A beta incubations resulted in a moderate attenuation of toxicity possibly due to a reduction in A beta-cell interactions caused by extensive lateral aggregation of the MAF-A beta complexes. Our results indicate that A beta is generally susceptible to proteoglycan-mediated aggregation and fibril formation. We also propose that the MAF model system may be useful in delineating these interactions and represent a means to develop and examine potential inhibitors of the proteoglycan effects.

A clinical and laboratory evaluation of immune serum globulin from donors with a history of hepatitis: attempted prevention of post-transfusion hepatitis.

A controlled trial of passive immunization for prevention of post-transfusion viral hepatitis was carried out in order to determine whether effective levels of antibody were present in the "convalescent" immune serum globulin used in the study. This globulin was prepared selectively from plasma of donors giving a history of overt viral hepatitis two or more years earlier. The proportion of contributors to the globulin who had B hepatitis was unknown but the final product contained a low titer of antibody to the surface antigen of hepatitis B virus (anti-HBs). The failure of 20 ml of immune serum globulin to reduce the incidence of type B post-transfusion hepatitis (7/93) below that of placebo-treated controls (8/102) was not unexpected in view of the globulin's low titer of anti-HBs. However, more than two thirds of the post-transfusion cases were not type B and were as plentiful among globulin recipients (17/93) as among controls (17/102). Although some of the donors from whom the immune serum globulin was obtained may once have had the same type(s) of hepatitis as the non-B cases currently observed in transfusion recipients, the globulin apparently did not contain enough specific antibody to confer protection in the dose schedule tested.