guanosine-triphosphate and Burkitt-Lymphoma

The basis for mutations at A:T base pairs in immunoglobulin hypermutation and defining how AID interacts with the DNA of the immunoglobulin locus are major aspects of the immunoglobulin mutator mechanism where questions remain unanswered. Here, we examined the pattern of mutations generated in mice deficient in various DNA repair proteins implicated in A:T mutation and found a previously unappreciated bias at G:C base pairs in spectra from mice simultaneously deficient in DNA mismatch repair and uracil DNA glycosylase. This suggests a strand-biased DNA transaction for AID delivery which is then masked by the mechanism that introduces A:T mutations. Additionally, we asked if any of the known components of the A:T mutation machinery underscore the basis for the paucity of A:T mutations in the Burkitt lymphoma cell lines, Ramos and BL2. Ramos and BL2 cells were proficient in MSH2/MSH6-mediated mismatch repair, and express high levels of wild-type, full-length DNA polymerase eta. In addition, Ramos cells have high levels of uracil DNA glycosylase protein and are proficient in base excision repair. These results suggest that Burkitt lymphoma cell lines may be deficient in an unidentified factor that recruits the machinery necessary for A:T mutation or that AID-mediated cytosine deamination in these cells may be processed by conventional base excision repair truncating somatic hypermutation at the G:C phase. Either scenario suggests that cytosine deamination by AID is not enough to trigger A:T mutation, and that additional unidentified factors are required for full spectrum hypermutation in vivo.

Topics: Adenosine Triphosphate; Animals; Burkitt Lymphoma; Cell Line, Tumor; Cytidine Deaminase; Cytidine Triphosphate; DNA Mismatch Repair; DNA Repair Enzymes; Guanosine Triphosphate; Humans; Mice; Mutation; Nucleotides; Somatic Hypermutation, Immunoglobulin; Thymine Nucleotides

Computer-assisted structure analysis indicated (+)-discodermolide, a polyhydroxylated alkatetraene lactone marine natural product, was an antimitotic compound, and we confirmed this prediction. Previous work had shown an accumulation of discodermolide-treated cells in the G2/M portion of the cell cycle, and we have now found that discodermolide arrests Burkitt lymphoma cells in mitosis. Discodermolide-treated breast carcinoma cells displayed spectacular rearrangement of the microtubule cytoskeleton, including extensive microtubule bundling. Microtubule rearrangement that occurred with 10 nM discodermolide required 1 microM taxol. Discodermolide had equally impressive effects on tubulin assembly in vitro. Near-total polymerization occurred at 0 degree C with tubulin plus microtubule-associated proteins (MAPs) under conditions in which taxol at an identical concentration was inactive. Without MAPs and/or without GTP, tubulin assembly was also more vigorous with discodermolide than with taxol under every reaction condition examined. Discodermolide-induced polymer differed from taxol-induced polymer in that it was completely stable at 0 degree C in the presence of high concentrations of Ca2+. In a quantitative assay designed to select for agents more effective than taxol in inducing assembly, discodermolide had an EC50 value of 3.2 microM versus 23 microM for taxol.

Topics: Alkanes; Animals; Antineoplastic Agents; Breast Neoplasms; Burkitt Lymphoma; Calcium; Carbamates; Cell Division; Cell Line; Dose-Response Relationship, Drug; Female; Fluorescent Antibody Technique, Indirect; Guanosine Triphosphate; Humans; Kinetics; Lactones; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Paclitaxel; Porifera; Pyrones; Time Factors; Tubulin; Tumor Cells, Cultured

In the present work, anti-G protein antibodies were introduced inside streptolysin permeabilized O interleukin-2-activated natural killer (IANK) cells. Successful entry of the antibodies was determined by flow cytometry and fluorescence microscopy. Permeabilized cells showed typical large granular lymphocyte morphology and remained functional, significantly lysing both NK-sensitive K562 cells and NK-resistant/IANK-sensitive RAJI target cells. This method was utilized to study the effect of anti-G protein antibodies on the functional activities of IANK cells. Anti-Gs antibody inhibited IANK cell killing of RAJI but not of K562 target cells. Further analysis showed that K562 and RAJI cells enhance the binding of guanosine 5'-O-(thiotriphosphate) to IANK cell membranes, and increase the hydrolysis of [32P]GTP in these membranes. Immunoblot analysis showed that K562 and RAJI cells induce the release of alpha o, but not alpha i, alpha s, or alpha q.11 from IANK cell membranes. Cumulatively, these data indicate that putative receptors recognizing K562 or RAJI target cells are coupled to Go in IANK cells, however, only Gs seems to be coupled to receptors recognizing RAJI target cells. Our results point out the importance of Gs protein as a mediator of cellular cytotoxicity of the anti-tumor effector cells.

Topics: Animals; Antibodies; Bacterial Proteins; Burkitt Lymphoma; Cell Line; Cell Membrane Permeability; Cytotoxicity, Immunologic; Flow Cytometry; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Immunoglobulin G; Interleukin-2; Killer Cells, Natural; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphocyte Activation; Rabbits; Streptolysins; Tumor Cells, Cultured

To further study the mechanisms by which surface Ig triggering activates the inositol phospholipid signaling pathway, we have used B cells from chronic lymphocytic leukemia patients which, as previously described, display two patterns of response upon sIg cross-linking: in one group this cross-linking induces an inositol phosphate release, an intracellular free Ca2+ concentration elevation and a subsequent cell proliferation; in a second group none of these events occur although there is an increased class II Ag expression following anti-mu stimulation as in the first group. We have been able to demonstrate that the phosphatidyl inositol specific phospholipase C (PI-PLC) can be activated in permeabilized B cells from the first group by direct stimulation, with GPT gamma S, of a guanine nucleotide binding (G) protein. In addition, since anti-mu + GTP gamma S stimulate an increased inositol phosphate production in these cells, this suggests that surface Ig cross-linking activates PI-PLC via a G protein. However, in cells from the second group no inositol phosphate is released after GTP gamma S stimulation although PI-PLC can be directly activated by high Ca2+ concentrations. This reflects in these cells, an interruption of the signaling cascade sIg/G protein/PI-PLC at the level of the G protein or at the G protein/PI-PLC coupling. In cells from both groups PMA treatment, which is known to alter phosphatidyl inositol metabolism in B cells, completely inhibits PI-PLC activation even by high Ca2+ concentrations. These studies show that the phosphatidyl inositol-dependent signaling cascade after surface Ig triggering can be altered at different levels in B cells.

Topics: B-Lymphocytes; Burkitt Lymphoma; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; HLA-D Antigens; Humans; In Vitro Techniques; Inositol Phosphates; Lymphocyte Activation; Phorbol Esters; Phosphatidylinositols; Receptor Aggregation; Receptors, Antigen, B-Cell; Signal Transduction; Thionucleotides; Tumor Cells, Cultured; Type C Phospholipases