sodium-dodecyl-sulfate and Burkitt-Lymphoma

Many p53 functions require p53 transport into the nucleus. Mutant p53 also generally accumulates in the nucleus of transformed or neoplastic cells. However, examples of cytoplasmic accumulation of wild-type or mutant p53 have also been reported. Various explanations have been provided for defective nuclear localization. Here we propose a novel example of cytoplasmic p53 localization which occurs in cells showing gene amplification and appears to be due to the formation of stable p53 multimers. We studied a methotrexate-resistant Chinese hamster cell line (MTX M) carrying amplified dihydrofolate reductase genes and derived from a cell line with p53 nuclear accumulation. MTX M showed cytoplasmic p53 localization and, on immunoblots, several extra bands in the high molecular weight region, besides the expected 53 kDa band. p53 localization and the appearance of high molecular weight bands appeared to be correlated with the degree of DNA amplification. However, amplification of dihydrofolate reductase itself was not involved. Changing the p53 phosphorylation status quantitatively influenced the formation of high molecular weight bands. Cell fusion experiments demonstrated that p53 cytoplasmic localization in MTX M is a dominant phenotype. This result suggests that the defect causing lack of nuclear localization in this cell line does not reside in the nucleus. In the cytoplasm of MTX M and of wild-type/MTX M heterodikaryons p53 gives rise to protein complexes that are unable to re-enter the nucleus. The formation of such protein complexes is dependent on the amplification of an unknown gene product.

Topics: 3T3 Cells; Animals; Antimetabolites, Antineoplastic; Burkitt Lymphoma; Cell Line, Transformed; Cell Nucleus; Cricetinae; Cricetulus; Cytoplasm; Dithiothreitol; DNA; Drug Resistance, Neoplasm; Gene Amplification; Humans; Immunoblotting; Methotrexate; Mice; Phenotype; Phosphorylation; Precipitin Tests; Sodium Dodecyl Sulfate; Tetrahydrofolate Dehydrogenase; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The CD19+ CD38+ human Burkitt's lymphoma cell line Ramos grows aggressively when injected intravenously (i.v.) into severe combined immunodeficient (SCID) mice, killing 100% of animals within a 33-42 day period with widely disseminated disease. Treatment commencing 7 days after i.v. injection of Ramos cells, with 3 doses of an anti-CD19 immunotoxin (IT; BU12-SAPORIN) or an anti-CD38IT (OKT10-SAPORIN) led to a significant prolongation of survival compared with sham-treated controls; the anti-CD38 IT gave the greatest prolongation of survival, but all treated animals eventually succumbed to disease. When both ITs were used in combination at equivalent dose levels, the therapeutic outcome was significantly improved over that obtained for single IT therapy, with 20% of animals surviving disease-free to 300 days. When anti-CD38 IT was given in combination with anti-CD19 antibody there was no therapeutic improvement over anti-CD38 IT used alone. However, when anti-CD19 IT was given in combination with CD38 antibody, a significant prolongation of survival ensued over that obtained with anti-CD19 IT alone, though this was not as significantly pronounced as that obtained when both ITs were used in combination and was only as good as the survival obtained with OKT10 antibody used alone. CD19 and CD38 are expressed on the surface of the vast majority of B-cell lymphoma and common acute lymphoblastic leukaemia cells, and our findings provide a sound rationale for a combination immunotoxin trial in these diseases directed against both these target molecules.

Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Animals; Antibodies; Antigens, CD; Antigens, CD19; Antigens, Differentiation; Antigens, Differentiation, B-Lymphocyte; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Burkitt Lymphoma; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immunotoxins; Male; Membrane Glycoproteins; Mice; Mice, SCID; N-Glycosyl Hydrolases; Neoplasm Proteins; Neoplasm Transplantation; Plant Proteins; Ribosome Inactivating Proteins, Type 1; Saporins; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

The immunotoxin BU12-SAPORIN was constructed by covalently coupling the single-chain ribosome-inactivating protein saporin to the anti-CD19 monoclonal antibody BU12 via a disulphide linker using the heterobifunctional reagent SPDP. The immunoreactivity and specificity of BU12-SAPORIN was identical to that of unmodified native BU12 antibody. BU12-SAPORIN was selectively cytotoxic in vitro in a dose-dependent manner for the CD19+ human common acute lymphoblastic leukaemia (cALL) cell line NALM-6 but exhibited no toxicity for the CD19- T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2. The survival of severe combined immunodeficient (SCID) mice with disseminated NALM-6 leukaemia was significantly prolonged compared with sham-treated control animals by a course of therapy with BU12-SAPORIN but not with the irrelevant anti-CD7 immunotoxin HB2-SAPORIN. BU12-SAPORIN had no therapeutic effect in SCID mice with disseminated CD19- HSB-2 leukaemia. These preclinical studies have clearly demonstrated the selective cytotoxicity of BU12-SAPORIN for CD19+ target cells both in vitro and in vivo. This, taken together with the lack of expression of the CD19 molecule by any normal life-sustaining tissue and its ubiquitous and homogeneous expression by the majority of cALL and B-NHL cells, provides the rationale for undertaking a phase I trial of systemic therapy with BU12-SAPORIN.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD19; Antigens, Neoplasm; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; B-Lymphocytes; Burkitt Lymphoma; Drug Screening Assays, Antitumor; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immunotoxins; Leukemia, B-Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Mice, SCID; N-Glycosyl Hydrolases; Neoplasm Proteins; Neoplasm Transplantation; Plant Proteins; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Protein Synthesis Inhibitors; Ribosome Inactivating Proteins, Type 1; Saporins; Sodium Dodecyl Sulfate

Complement receptor activity for cell bound C3b and C3d was detected on plasma membrane fragments prepared by nitrogen cavitation from cultured human lymphoid cells. The activity of the membrane fragments reflected the activity of the whole cells in that cells which did not form rosettes (P3J and RPMI 4098) resulted in inactive membranes and cells with high rosette formation (NC37 and Raji) yielded highly active membrane fragments. Two test systems were devised to detect these receptor activities, namely a rosette inhibition and a hemagglutination assay. Solubilization of C3 receptors was accomplished by extraction of active plasma membrane fragments with 2 MKBr. Dissociation and reassociation experiments suggest C3b and C3d receptors to be highly complex molecular structures. It appears that these complement receptors on plasma membranes rely on both protein and lipid moieties for the expression of their activity.

Topics: Binding Sites; Burkitt Lymphoma; Butanols; Cell Fractionation; Cell Line; Cell Membrane; Cells, Cultured; Complement C3; Complement System Proteins; Deoxycholic Acid; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Ethanol; Hemagglutination Tests; HEPES; Humans; Immune Adherence Reaction; Leukemia, Myeloid; Lipids; Lymphocytes; Neoplasm Proteins; Polyethylene Glycols; Sodium Dodecyl Sulfate; Sucrose

Topics: Animals; Antigens, Neoplasm; Burkitt Lymphoma; Cell Line; Chromatography, Affinity; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Humans; Immune Sera; Iodine Radioisotopes; Leukemia; Leukemia, Myeloid; Mercaptoethanol; Molecular Weight; Papain; Rabbits; Sodium Dodecyl Sulfate; Urea

Topics: Burkitt Lymphoma; Cell Division; Cell Nucleus; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Humans; Lectins; Leukemia; Leukemia, Lymphoid; Leukocytes; Lymphocyte Activation; Lymphocytes; Molecular Weight; Peptide Hydrolases; Phosphoric Monoester Hydrolases; Phosphorus Radioisotopes; Proteins; Sodium Dodecyl Sulfate; Tritium

Topics: Acrylamides; Burkitt Lymphoma; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; DNA, Neoplasm; Electrophoresis; Genetic Code; Humans; In Vitro Techniques; Lectins; Leukemia; Leukemia, Lymphoid; Leukemia, Monocytic, Acute; Leukemia, Myeloid; Lymphocytes; Neoplasm Proteins; Nucleoproteins; Sodium Dodecyl Sulfate; Thymidine; Tritium

Cells from an established line of Burkitt lymphoma (Daudi) were enzymatically radioiodinated, and labeled Ig from the cell surface was isolated and studied. Subcellular fractionation of labeled cells confirmed that intracellular proteins from the cytoplasm are not iodinated by this method. Radioactive Ig was identified as monomeric (8S) IgM, and an average of 10(5) Ig molecules was found per cell. Ig molecules could be released from the plasma membrane by detergent lysis under nonreducing conditions indicating that attachment of Ig to the plasma membrane occurs via noncovalent interactions. The ratio of micro/L radioactivity in surface Ig was the same as that of total cellular Ig radioiodinated in solution suggesting that a large portion of the Fc fragment is not buried within the membrane. In contrast to the results obtained with cell surface Ig, most intracellular Ig was found as "free" micro- and L chains regardless of whether lysates were labeled with (125)I or cells were labeled with leucine-(3)H. The results indicate that only a small percentage of the total Ig of Daudi cells is associated with the cell surface and suggest that covalent assembly of Ig occurs at or near the time that the molecule becomes part of the plasma membrane. Similarities between cell surface Ig on normal splenic lymphocytes and Daudi cells suggest that the latter is a neoplasm of bone marrow-derived lymphocytes.

Topics: Acrylamides; Amides; Burkitt Lymphoma; Cell Fractionation; Cell Line; Cell Membrane; Electrophoresis; Humans; Immunity, Cellular; Immunoglobulins; Iodine Isotopes; Molecular Weight; Peptides; Precipitin Tests; Sodium Dodecyl Sulfate