globotriaosylceramide and Carcinoma

Shiga toxin is a bacterial toxin consisting of A and B subunits. Generally, the essential cytotoxicity of the toxin is thought to be mediated by the A subunit, which possesses RNA cleavage activity and thus induces protein synthesis inhibition. We previously reported, however, that the binding of the Shiga toxin 1-B subunit to globotriaosyl ceramide, a functional receptor for Shiga toxin, induces intracellular signals in a manner that is dependent on glycolipid-enriched membrane domains, or lipid rafts. Although the precise role of this signaling mechanism is not known, here we report that Shiga-toxin-mediated intracellular signals induce cytoskeleton remodeling in ACHN cells derived from renal tubular epithelial carcinoma. Using confocal laser scanning microscopy, we observed that Shiga toxin 1-B treatment induces morphological changes in ACHN cells in a time-dependent manner. In addition, the morphological changes were accompanied by the redistribution of a number of proteins, including actin, ezrin, CD44, vimentin, cytokeratin, paxillin, FAK, and alpha- and gamma-tubulins, all of which are involved in cytoskeletal organization. The transient phosphorylation of ezrin and paxillin was also observed during the course of protein redistribution. Experiments using inhibitors for a variety of kinases suggested the involvement of lipid rafts, Src family protein kinase, PI 3-kinase, and RHO-associated kinase in Shiga toxin 1-B-induced ezrin phosphorylation. Shiga toxin 1-B-induced cytoskeletal remodeling should provide an in vitro model that can be used to increase our understanding of the pathogenesis of Shiga-toxin-mediated cell injury and the role of lipid-raft-mediated cell signaling in cytoskeletal remodeling.

Topics: Actins; Carcinoma; Cell Line; Cell Line, Tumor; Cytoskeletal Proteins; Cytoskeleton; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Hyaluronan Receptors; Immunoblotting; Immunohistochemistry; Kidney; Kidney Neoplasms; Lipid Metabolism; Microscopy, Confocal; Microscopy, Fluorescence; Models, Biological; Paxillin; Phosphoproteins; Phosphorylation; Protein Binding; Protein-Tyrosine Kinases; RNA; Shiga Toxin; Shiga Toxin 1; Signal Transduction; Time Factors; Trihexosylceramides; Tubulin; Vimentin

The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1), targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. CD77 and/or SLT-1 binding was detected by flow cytometry and immunocytochemistry on lymphoma and breast cancer cells recovered from biopsies of primary human cancers as well as on B cells or plasma cells present in blood/bone marrow samples of multiple myeloma patients. Breast cancer cell lines also expressed receptors for the toxin and were sensitive to SLT-1. Treatment of primary B lymphoma, B-cell chronic lymphocytic leukemia, and myeloma B or plasma cells with SLT-1-depleted malignant B cells by 3- to 28-fold, as measured by flow cytometry. Depletion of myeloma plasma cells was confirmed using a cellular limiting dilution assay followed by reverse transcriptase-polymerase chain reaction analysis of clonotypic IgH transcripts, which showed a greater than 3 log reduction in clonotypic myeloma cells after SLT-1 treatment. Receptors for the toxin were not detected on human CD34(+) hematopoietic progenitor cells (HPC). HPC were pretreated with a concentration of SLT-1 known to purge primary malignant B cells and cultured for 6 days. The number of HPC was comparable in toxin-treated and untreated cultures. HPC were functionally intact as well. Colony-forming units (CFU) were present at an identical frequency in untreated and SLT-1 pretreated cultures, confirming that CFU escape SLT-1 toxicity. The results suggest the ex vivo use of SLT-1 in purging SLT-1 receptor-expressing malignant cells from autologous stem cell grafts of breast cancer, lymphoma, and myeloma patients.

Topics: Antibodies, Monoclonal; B-Lymphocytes; Bacterial Toxins; Biomarkers; Biomarkers, Tumor; Blood Cells; Bone Marrow Cells; Bone Marrow Purging; Breast Neoplasms; Carcinoma; Cell Separation; Cells, Cultured; Colony-Forming Units Assay; Female; Flow Cytometry; Genes, Immunoglobulin; Glycolipids; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Immunoglobulin Heavy Chains; Lymphoma, B-Cell; Lymphoma, Follicular; Male; Multiple Myeloma; Neoplasm Proteins; Neoplastic Stem Cells; Organ Specificity; Plasma Cells; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; Shiga Toxin 1; Transplantation, Autologous; Trihexosylceramides; Tumor Cells, Cultured; Tumor Stem Cell Assay