sepharose and Lymphoma--Non-Hodgkin

Bifunctional alkylating agents, such as melphalan, are widely used in the treatment of hematological malignancies. The effects of these drugs on particular types of hematological cells and the causes of treatment failure are poorly understood. The aim of this work was to establish an ability to measure the extent to which melphalan reacts with the DNA of individual tumor cells, thereby creating new possibilities for molecular pharmacological studies on clinical samples. A novel approach for staining drug-DNA adducts is described in which cells were embedded in agarose and then lysed. The DNA from each cell remained in an ideal state for quantitative immunofluorescent staining using a previously described monoclonal antibody. Immunofluorescence and DNA-Hoechst dye fluorescence were quantified using a cooled slow scan charge coupled device camera and image analysis procedures. Immunofluorescence of drug-treated cells from a human leukemia cell line was partially correlated with DNA content. Mean integrated immunofluorescence of 50 to 100 cells was dependent on drug concentration and was linearly related to adduct levels. In these cells and in chronic lymphocytic leukemia cells obtained from patients, there was considerable intercell heterogeneity in apparent adduct levels. This was also seen in peripheral blood mononuclear cells isolated from a patient after melphalan therapy.

Topics: Antineoplastic Agents, Alkylating; Bisbenzimidazole; DNA Adducts; DNA, Neoplasm; Feasibility Studies; Fluorescent Dyes; Hematopoietic Stem Cells; Humans; Image Processing, Computer-Assisted; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Melphalan; Neoplastic Stem Cells; Sepharose; Staining and Labeling; Tissue Embedding; Tumor Cells, Cultured

Topics: Animals; Cell Line; Chromatography, Affinity; Deoxyadenine Nucleotides; Lymphoma, Non-Hodgkin; Mice; Mutation; Ribonucleotide Reductases; Sepharose

Plastic dishes were coated with an agarose layer. The layer was modified by covalently binding proteins to it, using the CNBr-method. Cells were seeded on the dishes and the number of attached cells was evaluated. The specificity of the attachment was demonstrated by showing that cells, carrying specific membrane-bound immunoglobulins, attached only to the corresponding anti-immunoglobulins. This indicated that the method could be used for cell sorting. The attachment of cells to proteins was influenced by the amount of bound protein, incubation time, temperature and the degree of trypsinization. Most attached cells were viable for several days and when dying they detached. Detailed morphological and cytochemical analyses of the dynamics of attachment and cytoplasmic spreading on the chemically well-defined surfaces were possible using the new method.

Topics: Burkitt Lymphoma; Cell Adhesion; Cell Line; Concanavalin A; Glioma; Humans; Leukemia; Lymphoma, Non-Hodgkin; Neuroglia; Polylysine; Polysaccharides; Protamines; Protein Binding; Proteins; Sepharose