globotriaosylceramide and Leukemia

We have used two methods to evaluate the level of expression of Gb3Cer in several human leukaemia/lymphoma cell lines representative of the myeloid (K562, KG-1, HL-60, and lymphoid (Reh, Daudi, Raji, RPMI 8226, CCRF-CEM, MOLT-4) lineages blocked at varied stages of differentiation. TLC immunostaining of glycolipid extracts with a monoclonal antibody, 12-101, and FACS analysis with the same antibody were used to demonstrate that the expression of Gb3Cer in neoplastic myeloid and lymphoid cells is both lineage and differentiation dependent. As a possible control point in the regulated expression of Gb3Cer we have investigated the first committed step in the synthesis of globo series glycosphingolipids that involves UDP-Gal:LacCer alpha (1,4)-galactosyltransferase (alpha 1,4GalT). We present the first characterization of this enzyme in a human myeloid cell line using an ELISA-based assay, which was subsequently used to measure alpha 1,4GalT activity in the human leukaemia/lymphoma cell lines. In general, there is a positive correlation between the levels of endogenous Gb3Cer and the level of the alpha 1,4 GalT activity. However, in two cases (KG-1 and CCRF-CEM) the level of enzyme activity did not correspond to the level of Gb3Cer expression.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Cell Differentiation; Cell Line; Chromatography, Thin Layer; Flow Cytometry; Galactosyltransferases; Glycosphingolipids; HL-60 Cells; Humans; Leukemia; Lymphoma; Molecular Sequence Data; Monocytes; Trihexosylceramides; Tumor Cells, Cultured

The purpose of this study was to isolate mouse monoclonal antibodies (MAbs) recognizing Gal alpha 1-4Gal beta 1-4Glc (Pk, CD77), which has been described as a Burkitt's-lymphoma-associated antigen. Three IgGI MAbs reactive with Pk were developed using a synthetic glycoconjugate as immunogen and a filter immunoplaque screening assay. One MAb (PK67) was characterized by immuno-thin-layer chromatography, ELISA and competition assays using neutral glycolipids from various sources and a variety of carbohydrates and glycoproteins. Epitope analysis showed that all 3 carbohydrates moieties are required in PK67 binding. No cross-reactivity was observed with closely related carbohydrate structures, with the exception of Gal alpha 1-4Gal beta 1-4GlcNAc. However, the reactivity with this structure was several orders of magnitude lower than with Pk. Immunohistochemical analysis of fresh-frozen tissue specimens showed that Gb3 is widely expressed: prominent staining was observed in many normal tissues, e.g., kidney and gastric tissue and on capillary endothelial cells. In lymph nodes, very weak staining of a few B cells was observed. Flow cytometric analysis of hematopoietic neoplasms showed Gb3 expression on B-cell neoplasms, particularly those with committed B-cell phenotype indicating that, in addition to earlier reports of Gb3 expression on Burkitt's lymphoma, Gb3 is present on other committed B-cell neoplasms.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Carbohydrate Sequence; Carbohydrates; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Glycolipids; Glycoproteins; Humans; Hybridomas; Immunoenzyme Techniques; Leukemia; Lymphoma; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Neoplasms; Organ Specificity; Reference Values; Trihexosylceramides