• A B cell specific immediate early human gene is located on chromosome band 1q31 and encodes an alpha helical basic phosphoprotein.

      Newton, J S; Deed, Richard W; Mitchell, Erika L D; Murphy, J J; Norton, John D; Immunology Section, Kings College London, UK. (1993-11-16)
      We have determined the cDNA sequence of a human B cell specific, immediate early gene, designated 1R20, which is inducible in response to several B cell activation signals. The cDNA sequence predicts a 196 amino acid open reading frame comprising numerous highly basic residues and the predicted structure contains several potential alpha helical domains together with eight consensus protein phosphorylation sites. The 1R20 gene has been localised by fluorescence in situ hybridisation to chromosome band 1q31, a region known to be implicated in the pathogenesis of haemopoietic malignancies.
    • Distinct mechanisms for rescue from apoptosis in Ramos human B cells by signaling through CD40 and interleukin-4 receptor: role for inhibition of an early response gene, Berg36.

      Ning, Z Q; Norton, John D; Li, J; Murphy, J J; Infection and Immunity Research group, King's College London, GB. (1996-10)
      The role of interleukin-4 (IL-4) and CD40 signaling in negative regulation of apoptosis in human Ramos B cells induced in response to different agents was investigated. CD40 ligation protected cells from apoptosis induced by calcium ionophore through an initial, rapid and apparently Bcl-2-independent mechanism, associated with up-regulation of Bcl-XL. However, rescue from apoptosis induced by inhibition of macromolecular synthesis required several hours of prior stimulation with CD40 ligand/antibody and was accompanied by up-regulation of Bcl-2. In contrast, IL-4 did not up-regulate Bcl-2 or Bcl-XL and did not inhibit apoptosis induced by inhibitors of macromolecular synthesis. However, IL-4 did protect Ramos cells from apoptosis induced by calcium ionophore and this effect was accompanied by inhibition of ionophore-induced expression of an immediate early gene encoding a 36-kDa zinc-finger protein, Berg36. Antisense blockade of Berg36 expression partially inhibited ionophore-induced apoptosis to an extent commensurate with the level of IL-4 protection, implicating Berg36 function as a requirement for apoptosis induced through calcium signaling and as a target for IL-4 through which this cytokine inhibits apoptosis in Ramos B cells. These distinct mechanisms for rescue from apoptosis by CD40 and IL-4 may help explain the co-operative roles of these T cell-derived signals for B cell survival.
    • Early gene signalling-dependent and -independent induction of apoptosis in Ramos human B cells can be inhibited by over-expression of Bcl-2.

      Ning, Z Q; Norton, John D; Johnson, Diane; Murphy, J J; Division of Life Sciences, King's College London, UK. (1995-10-04)
      We have previously shown that calcium ionophore-induced apoptosis of Ramos human B cells is preceded by the induced expression of early response genes, implying a requirement for new gene expression in this mode of programmed cell death. We have found in the present studies that inhibitors of macromolecular synthesis, cycloheximide and actinomycin D, are also potent inducers of apoptosis in the same Ramos cell model. These drugs trigger apoptosis through apparently early gene signalling-independent pathways. Although different mechanisms for induction of apoptosis exist in Ramos cells, enforced over-expression of Bcl-2 protects cells from apoptosis induced in response to different agents, demonstrating that Bcl-2 blocks a final common pathway for programmed cell death in the Ramos cell model.
    • Early response gene signalling in bryostatin-stimulated primary B chronic lymphocytic leukaemia cells in vitro.

      Ning, Z Q; Hirose, Tohru; Deed, Richard W; Newton, J; Murphy, J J; Norton, John D; Division of Life Sciences, King's College London, U.K. (1996-10-01)
      The protein kinase C activator bryostatin induces differentiation and antagonizes the effects of tumour-promoting phorbol esters in a number of different cell types. We show here that bryostatin preferentially inhibits phorbol 12-myristate 13-acetate (PMA)-induced proliferation compared with differentiation in a number of different B chronic lymphocytic leukaemia (BCLL) cell populations examined. By using a panel of 11 early-response gene probes in Northern hybridization analysis, we found that the profile of genes induced in response to bryostatin and PMA was qualitatively similar and displayed comparable sensitivities to inhibition with the serine-threonine kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine hydrochloride (H7), consistent with common signalling through protein kinase C. However, the nuclear oncogene. c-myc, which was induced strongly in response to PMA treatment, was only marginally up-regulated by bryostatin. In addition, bryostatin selectively inhibited the magnitude of PMA-responsive induction of c-myc, to a degree commensurate with its antagonistic effects seen at the biological level. Finally, an anti-sense oligonucleotide blockade of c-myc inhibited PMA-induced proliferation but not the differentiation of BCLL cells, implicating this nuclear oncogene as an important determinant distinguishing PMA from bryostatin-coupled biological responses and also as a candidate third-messenger effector target for the anti-tumour effects of bryostatin.
    • Ectopic expression of Bcl-2, but not Bcl-xL rescues Ramos B cells from Fas-mediated apoptosis.

      Alam, M K; Davison, S; Siddiqui, N; Norton, John D; Murphy, J J; Infection and Immunity Research Group, Division of Life Sciences, King's College London, GB. (1997-12)
      The human Burkitt lymphoma Ramos B cell line can be induced to undergo apoptosis in response to a variety of different agents, including calcium ionophores, anti-immunoglobulin (Ig) and macromolecular synthesis inhibitors. In addition, following up-regulation of the Fas (CD95) surface receptor by CD40 ligation, these cells also become susceptible to apoptosis induction by Fas ligation. We have previously shown that protection from calcium ionophore- and macromolecular synthesis inhibitor-induced apoptosis by CD40 ligation is associated with a rapid up-regulation of Bcl-xL followed by a more moderate and delayed up-regulation of Bcl-2. We show here that overexpression of Bcl-xL, like Bcl-2, protects Ramos cells from apoptosis induction in response to calcium ionophore, anti-Ig and macromolecular synthesis inhibition. However, in contrast to Bcl-2, ectopic overexpression of Bcl-xL does not rescue from Fas-mediated apoptosis. Thus, in Ramos B cells, the Fas apoptotic pathway exhibits differential sensitivity to inhibition by Bcl-2 family members. These findings also suggest that CD40 signaling provides a switch which renders the cells susceptible to Fas-ligand mediated apoptosis through up-regulation of Fas whilst affording protection from anti-Ig-induced apoptosis through up-regulation of Bcl-xL.
    • An immediate early human gene encodes an Id-like helix-loop-helix protein and is regulated by protein kinase C activation in diverse cell types.

      Deed, Richard W; Bianchi, S M; Atherton, Graham T; Johnston, D; Santibanez-Koref, Mauro F; Murphy, J J; Norton, John D; CRC Department of Gene Regulation, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1993-03)
      Transcription factors characterized by the presence of a helix-loop-helix (HLH) domain play a central role in the regulation of cell growth/differentiation and tumorigenesis. We report here the cDNA sequence of a human early-response gene, designated HLH 1R21, encoding a 15-kDa HLH protein that lacks a basic, DNA-binding domain and which by a number of criteria appears to be the human homologue of mouse HLH 462. Like its murine counterpart, HLH 1R21 protein functions as an Id (inhibitor of DNA binding) transcription factor by inhibiting the binding of E2A-containing protein complexes to muscle creatine kinase E-box enhancer oligonucleotide in vitro. However HLH 1R21 does not inhibit the binding of HLH Max protein to a Max-binding oligonucleotide in vitro, indicating that it has limited promiscuity in its ability to antagonize the function of other HLH transcription factors. In addition, HLH 1R21 mRNA transcripts are regulated by phorbol ester treatment of a diverse range of human cell lines and, when overexpressed in mouse NIH3T3 cells, HLH 1R21 induces a morphologically transformed phenotype.
    • Multiple signaling pathways mediate anti-Ig and IL-4-induced early response gene expression in human tonsillar B cells.

      Murphy, J J; Norton, John D; Division of Life Sciences, King's College London, GB. (1993-11)
      We have analyzed the relationship between the signaling pathways coupled to surface immunoglobulin and interleukin (IL)-4 receptors in human B cells from the patterns of expression of a panel of phorbol ester-inducible early response genes (ERG) activated by anti-IgM and IL-4 stimulation in vitro. Anti-IgM stimulation led to the induction of all eleven ERG tested. Two of these, the proto-oncogene, c-fos and an anonymous ERG 1R20 were insensitive to protein kinase C (PKC) inhibition with the drug, staurosporine and retained inducibility after down-regulation of PKC activity by purging with phorbol ester. These observations are consistent with previous data showing anti-IgM signaling through both PKC-dependent and PKC-independent pathways. c-fos and 1R20 were also the only ERG inducible in response to IL-4 stimulation and whilst ionomycin induced only c-fos, dibutyryl cyclic adenosine monophosphate stimulation led to induction of both c-fos and 1R20. These observations lend support to a role for the adenylate cyclase pathway being important for coupling of IL-4-generated signals to B cells responses. None of the anti-IgM-responsive ERG was further induced when B cells were co-stimulated with a combination of anti-IgM and IL-4, suggesting that the signaling cascades from these two agents are integrated downstream of third messenger pathways to synergistically promote B cell proliferation.
    • Phorbol ester induction of early response gene expression in lymphocytic leukemia and normal human B-cells

      Murphy, J J; Norton, John D; Infection and Immunity Research Group, Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH UK (1993)