pervanadate and Lymphoma--B-Cell

Bruton's tyrosine kinase (BTK) was previously demonstrated to be a mediator of oxidative stress-induced apoptosis in irradiated neoplastic B-cells and B-cell precursors. Defective BTK expression in leukaemic B-cell precursors from infants with t(4;11) acute lymphoblastic leukaemia has been associated with radiation resistance. The present study examined whether BTK mediates apoptosis during oxidative stress by interfering with the anti-apoptotic function of signal transducer and activator of transcription 3 (STAT3). BTK physically associated with and tyrosine phosphorylated STAT3; this association was promoted by pervanadate (PV)-induced oxidative stress. The BTK/STAT3 interaction appeared to prevent STAT3 response to oxidative stress, because PV-induced STAT3 activation was markedly enhanced in DT40 chicken lymphoma B-cells that were rendered BTK-deficient by targeted disruption of the btk gene as well as in BTK-deficient RAMOS-1 human lymphoma B-cells. These BTK-deficient cells were highly resistant to oxidative stress-induced apoptosis triggered by PV treatment. Reconstitution of BTK-deficient DT40 cells with wild-type human BTK gene eliminated the amplification of the STAT3 response and restored the PV-induced apoptotic signal. Similarly, while the BTK-positive NALM-6 human leukaemic B-cell precursor cell line showed no STAT3 activation after PV treatment and was exquisitely sensitive to PV-induced apoptosis, PV failed to induce apoptosis in BTK-deficient RAMOS-1 human lymphoma B-cells that showed a robust STAT3 response. These results provide unprecedented biochemical and genetic evidence for a unique mode of cross-talk that occurs between BTK and STAT3 pathways during oxidative stress, whereby BTK may trigger apoptosis via negative regulation of the anti-apoptotic STAT3 activity.

Topics: Agammaglobulinaemia Tyrosine Kinase; Animals; Apoptosis; Chickens; Enzyme Inhibitors; Female; Humans; Leukemia, B-Cell; Lymphoma, B-Cell; Neoplasm Proteins; Oxidative Stress; Phosphorylation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Recombinant Proteins; STAT3 Transcription Factor; Tumor Cells, Cultured; Vanadates

Exposure of wild-type DT40 lymphoma B cells or Bruton's tyrosine kinase (BTK)-deficient DT40 cells reconstituted with the human btk gene to a 1-gauss 60-Hz electromagnetic field (EMF) has been reported to rapidly increase inositol 1,4,5-trisphosphate (Ins 1,4, 5-P3) production (1,2). Here we have used BTK-deficient DT40 B cells reconstituted with the human btk gene to evaluate the reproducibility of these findings. An experimental design with blinded exposures and anti-IgM treatment to induce Ins 1,4,5-P3 production as a positive control, showed no significant effect of a 1-gauss 60-Hz EMF on Ins 1,4,5-P3 production. Because recent work has shown that the activation of BTK was required for EMF-responsiveness (2), we also evaluated the reproducibility of this finding in wild-type DT40 cells. BTK was activated in a dose- and time-dependent manner by treatment with the tyrosine phosphatase inhibitor pervanadate. However, the ability to detect BTK activation, as measured by increased autophosphorylation by immune complex kinase assay, was dependent on the kinase buffer. Using cells from the original investigators, no evidence was obtained to support the hypothesis that exposure to a 1-gauss 60-Hz EMF had a causal effect on protein-tyrosine kinase activities affecting Ins 1,4,5-P3 production.

Topics: Agammaglobulinaemia Tyrosine Kinase; Antibodies; Electromagnetic Fields; Enzyme Activation; Enzyme Induction; Humans; Immunoglobulin M; Inositol 1,4,5-Trisphosphate; Lymphoma, B-Cell; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Vanadates