B-cell precursor (BCP) leukemia is the most common form of childhood cancer and the second most common form of acute leukemia in adults. Human BCP leukemia was treated in a severe combined immunodeficient mouse model by targeting of the tyrosine kinase inhibitor Genistein (Gen) to the B cell-specific receptor CD19 with the monoclonal antibody B43. The B43-Gen immunoconjugate bound with high affinity to BCP leukemia cells, selectively inhibited CD19-associated tyrosine kinases, and triggered rapid apoptotic cell death. At less than one-tenth the maximum tolerated dose more than 99.999 percent of human BCP leukemia cells were killed, which led to 100 percent long-term event-free survival from an otherwise invariably fatal leukemia. The B43-Gen immuno-conjugate might be useful in eliminating leukemia cells in patients who have failed conventional therapy.
Functional impairment and selective depletion of CD4+ T cells, the hallmark of AIDS, are at least partly caused by human immunodeficiency virus (HIV-1) type 1 binding to the CD4 molecule and infecting CD4+ cells. It may, therefore, be of therapeutic value to target an antiviral agent to CD4+ cells to prevent infection and to inhibit HIV-1 production in patients' CD4+ cells which contain proviral DNA. We report here that HIV-1 replication in normal primary CD4+ T cells can be inhibited by pokeweed antiviral protein, a plant protein of relative molecular mass 30,000, which inhibits replication of certain plant RNA viruses, and of herpes simplex virus, poliovirus and influenza virus. Targeting pokeweed antiviral protein to CD4+ T cells by conjugating it to monoclonal antibodies reactive with CD5, CD7 or CD4 expressed on CD4+ cells, increased its anti-HIV potency up to 1,000-fold. HIV-1 replication is inhibited at picomolar concentrations of conjugates of pokeweed antiviral protein and monoclonal antibodies, which do not inhibit proliferation of normal CD4+ T cells or CD4-dependent responses. These conjugates inhibit HIV-1 protein synthesis and also strongly inhibit HIV-1 production in activated CD4+ T cells from infected patients.
Very little is known regarding the effects of ionizing radiation on cytoplasmic signal transduction pathways.Here, we show that ionizing radiation induces enhanced tyrosine phosphorylation of multiple substrates in human B-lymphocyte precursors. This response to ionizing radiation was also observed in cells pretreated with vanadate, a potent proteintyrosine-phosphatase (PTPase) inhibitor, and phosphotyrosyl [Vals]angiotensin II phosphatase assays showed no decreased PTPase activity in irradiated cells. Thus, enhanced tyrosine phosphorylation in irradiated B-lymphocyte precursors is not triggered by inhibition of total cellular PTPase activity. Immune-complex kinase assays using anti-phosphotyrosine antibodies demonstrated enhanced protein-tyrosine kinase (PTK) activity in the immunoprecipitates from irradiated cells, and the PTK inhibitors genistein and herbimycin effectively prevented radiation-induced tyrosine phosphorylation. Immune-complex kinase assays on irradiated and unirradiated B-lymphocyte precursors using antibodies prepared against unique amino acid sequences of pv9nf, p56/p53'Yn, p55""', and p561ck demonstrated that these Src-family tyrosine kinases were not the primary PTKs responsible for enhanced tyrosine kinase activity in the anti-phosphotyrosine antibody immunoprecipitates or for enhanced tyrosine phosphorylation of multiple substrates. Thus, our fmdings favor the hypothesis that ionizing radiation induces enhanced tyrosine phosphorylation in B-lymphocyte precursors by stimulation of as yet unidentified PTKs. Tyrosine phosphorylation appears to be an important proximal step in radiationinduced apoptosis and clonogenic cell death because inhibition of PTK prevents DNA fragmentation and loss of clonogenicity of irradiated B-lymphocyte precursors. Since PTKs play myriad roles in the regulation of cell function and proliferation, the activation of a PTK cascade, as detailed in this report, may explain some of the pleiotropic effects of ionizing radiation on cellular functions of B-lymphocytes and their precursors.The molecular mechanism by which ionizing radiation inhibits and destroys mammalian cells has been widely explored but not precisely deciphered (1, 2). Protein-tyrosine kinases (PTKs) participate and likely play pivotal roles in initiation of signal cascades that affect proliferation and survival of human B-lymphocyte precursors (3-7). The purpose of this study was to examine the effects of ionizing radiation on PTKs in human B-lymphocyte precursors at discrete developmental stages of B-cell ontogeny.MATERIALS AND METHODS Patient Material and Cell Lines. We used the fetal liver pro-B cell line FL112, the pre-pre-B cell line Reh, the pre-B cell line Nalm-6, and the early B/Burkitt lymphoma cell lines Daudi, Ramos, and Ramos-1 (a subclone of Ramos). The immunophenotypic and genotypic features and the radiation sensitivity of these human B-lymphocyte precursor cell lines were detailed in a previous report (8). We also used primary bone marrow blasts from a pre-pre-B acute lymphoblas...
Exposure of human B-cell precursors (BCP) to ionizing radiation results in cell cycle arrest at the G2-M checkpoint as a result of inhibitory tyrosine phosphorylation of p34cdc2 . Here, we show that ionizing radiation promotes physical interactions between p34cdc2 and the Src family protein-tyrosine kinase Lyn in the cytoplasm of human BCP leading to tyrosine phosphorylation of p34cdc2. Lyn kinase immunoprecipitated from lysates of irradiated BCP as well as a full-length glutathione S-transferase (GST)-Lyn fusion protein-phosphorylated recombinant human p34cdc2 on tyrosine 15. Furthermore, Lyn kinase physically associated with and tyrosine-phosphorylated p34cdc2 kinase in vivo when co-expressed in COS-7 cells. Binding experiments with truncated GST-Lyn fusion proteins suggested a functional role for the SH3 rather than the SH2 domain of Lyn in Lyn-p34cdc2 interactions in BCP. The first 27 residues of the unique amino-terminal domain of Lyn were also essential for the ability of GST-Lyn fusion proteins to bind to p34cdc2 from BCP lysates. Ionizing radiation failed to cause tyrosine phosphorylation of p34cdc2 or G2 arrest in Lyn kinase-deficient BCP, supporting an important role of Lyn kinase in radiation-induced G2 phase-specific cell cycle arrest. Our findings implicate Lyn as an important cytoplasmic suppressor of p34cdc2 function.
Extensive immunologic surface marker analyses and binding competition assays demonstrated that B43 monoclonal antibody (MoAb) is a new member of the CD19 cluster that recognizes the same surface epitope as several other anti-CD19 MoAbs. We used B43 MoAb to test for CD19 expression on neoplastic cells from 340 leukemia and 151 malignant lymphoma patients and on nonneoplastic cells in normal lymphohematopoietic and nonlymphohematopoietic tissues. Our study more than doubles the total number of cases with classified hematologic malignancies that have been examined for CD19 antigen expression. The data presented confirm that CD19 is the most reliable B lineage surface marker and support our view that this B lineage-restricted surface determinant may be an important functional receptor. Our findings provide unique and direct evidence that (a) CD19 is expressed on leukemic B lineage lymphoid progenitor cells freshly obtained from B lineage acute lymphoblastic leukemia patients but not on normal myeloid, erythroid, megakaryocytic, or multilineage bone marrow progenitor cells; (b) ligation of CD19 with B43 MoAb induces sustained increases in [Ca2+]i when crosslinked and inhibits high-molecular weight B cell growth factor (HMW-BCGF)-induced proliferation of activated B cells without affecting their low- molecular weight B cell growth factor (LMW-BCGF) response; therefore CD19 may be a unique signal receptor; (c) HMW-BCGF and LMW-BCGF augment expression of CD19, which suggests that CD19 and BCGF receptors may be under coordinate regulatory control; (d) approximately two million B43 MoAb molecules per cell can be bound to target B lineage lymphoma cells with a Ka of 1.9 x 10(8)/mol/L; (e) CD19 can undergo B43 MoAb-induced internalization; and (f) the opportunity is thus provided for using anti-CD19 MoAb to deliver toxins to B lineage neoplastic cells for more effective treatment of high-risk leukemia/lymphoma patients.
We have previously observed that ionizing radiation induces tyrosine phosphorylation in human B-lymphocyte precursors by stimulation of unidentified tyrosine kinases and this phosphorylation is substantially augmented by vanadate. Ionizing radiation generates reactive oxygen intermediates (ROI). Because H2O2 is a potent ROI generator that readily crosses the plasma membrane, we used H2O2 to examine the effects of ROI on signal transduction. We now provide evidence that the tyrosine kinase inhibitor herbimycin A and the free radical scavenger N- acetyl-cysteine inhibit both radiation-induced and H2O2-induced activation of NF-kappa B, indicating that activation triggered by ROI is dependent on tyrosine kinase activity. H2O2 was found to stimulate Ins-1,4,5-P3 production in a tyrosine kinase-dependent manner and to induce calcium signals that were greatly augmented by vanadate. The synergistic induction of tyrosine phosphorylation by H2O2 plus vanadate included physiologically relevant proteins such as PLC gamma 1. Although treatment of cells with H2O2 alone did not affect the activity of src family kinases, treatment with H2O2 plus vanadate led to activation of the p56lck and p59fyn tyrosine kinases. The combined inhibition of phosphatases and activation of kinases provides a potent mechanism for the synergistic effects of H2O2 plus vanadate. Induction of tyrosine phosphorylation by ROI may thus lead to many of the pleiotropic effects of ROI in lymphoid cells, including downstream activation of PLC gamma 1 and NF-kappa B.
Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme specific to lysine biosynthesis in plants, was In plants and bacteria, DHPS2 catalyzes the first step specific to lysine synthesis in the general pathway for biosynthesis of aspartate-derived amino acids including threonine, isoleucine, and methionine (2). DHPS isolated from plants is feedback inhibited by relatively low concentrations of lysine, indicating that subcellular end product concentrations contribute to regulation of DHPS activity and metabolite flux through the lysine-specific branch. This potential regulatory role in lysine synthesis has attracted interest to DHPS as a target for selection of feedback-resistant mutants (17) This study was initiated to obtain purified maize DHPS for physical characterization and for kinetic and inhibitor studies. We obtained highly purified DHPS from fully expanded maize leaf blades and cell suspension cultures with 23 to 25% recovery. The native enzyme is composed of four subunits with mol wt of approximately 38,000 and is subject to substrate inhibition by high ASA concentrations (>2 mM) and to feedback inhibition by low lysine concentrations (<100 ltM).A partial N-terminal amino acid sequence of the purified DHPS was also used to corroborate the nucleotide sequence of a cDNA clone for maize DHPS (8). MATERIALS AND METHODS Plant MaterialBlack Mexican Sweet maize (Zea mays L.) cell suspension cultures were maintained on a modified Murashige and Skoog medium by a 1:20 dilution into fresh liquid medium every 7 d (5). Cells were harvested during mid-log phase 5 d after subculture. Plants of inbred line A619 and two homozygous mutant lines, Ask-LTJ9 (12) and Ask2-LT20 (4), were grown in the field nursery at St. Paul, MN, in 1989. The Ask-LTJ9 and Ask2-LT20 alleles are altered forms of the aspartate kinase structural genes (Ask and Ask2) which result in reduced feedback inhibition of aspartate kinase by lysine (7). The two or three youngest, fully expanded leaves were harvested from plants at the five-leaf stage.
Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately flked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lympbocyte precursors, irradiation with -rays leads to (i) stimulation of phosphatidylinosltol turnover; (i) downstream activation by covalent modification of multiple swine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor KB. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase hiitors genistein and herbimycin A. Thus, tyrosine plosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation.Weichselbaum and colleagues (1, 2) have proposed that irradiation initiates a cascade of cytoplasmic signaling events in mammalian cells. In particular, ionizing radiation stimulates the transcription of immediate early response genes that encode transcriptional factors, by activation of a protein kinase C (PKC)-dependent cytoplasmic signaling pathway (1, 2). However, radiation-induced signaling events proximal to PKC activation require further evaluation.Protein-tyrosine kinases (PTKs) are key participants in the initiation of signal cascades that affect proliferation and survival of human B-lymphocyte precursors (3, 4). We have recently shown that ionizing radiation stimulates several PTlKs in human B-lymphocyte precursors, including the Src family tyrosine kinases p59fyn and p55bIk, leading to enhanced tyrosine phosphorylation of multiple substrates and triggering apoptosis (5). Since PTKs play myriad roles in the regulation of cell function and proliferation (6, 7), the activation of a PIK cascade and tyrosine phosphorylation may explain the pleiotropic effects of ionizing radiation on cellular functions.The present study expands earlier work by specifically examining the role of tyrosine phosphorylation in radiationinduced activation of the PKC pathway in human B-lymphocyte precursors at discrete developmental stages of B-cell ontogeny. Notably, the tyrosine kinase inhibitors herbimycin and genistein effectively inhibited radiation-induced activation of PKC (PK76) and PKC-dependent seine kinases PK55 and PK50. Furthermore, the activation of nuclear factor KB (NF-KB) in irradiated B-lymphocyte precursors was abrogated by pretreatment with herbimycin or genistein. Our results indicate that radiation-induced activation of the PKC signaling cascade leads to downstream activation of multiple serine-specific protein kinases, and activation of NF-KB. The evidence provided reveals that these events are triggered by radiation-induced stimulation of tyrosine-specific protein kinases. MATERIALS AND METHODSPatient Material and Cell Lines. We used the CD19+CD10+ CasIgM-pre-pre-B cell line REH, the CD19+CD10+ CsIgM...
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