RESUMO
Nuclear factor-kappaB (NF-kappaB) activity affects cell survival and determines the sensitivity of cancer cells to cytotoxic agents as well as to ionizing radiation. Preventing the protective function of NF-kappaB may result in chemo- and radio-sensitization of cancer cells. Therefore, NF-kappaB has emerged as one of the most promising molecular targets in rational drug design efforts of translational cancer research programs.
Assuntos
Regulação da Expressão Gênica , NF-kappa B/metabolismo , Neoplasias/terapia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Desenho de Fármacos , Regulação Viral da Expressão Gênica , Humanos , Modelos Biológicos , Radiação Ionizante , Transdução de Sinais , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Modulation of Ras function may provide a novel means by which cancer cells with oncogenic mutations can be sensitized to chemotherapeutic or radiotherapeutic regimens. Moreover, cancer cells without ras oncogene mutations can also be eliminated by compounds that interfere with the mevalonate pathway, which is more fundamental to mitogenesis because it allows the synthesis of sterol and nonsterol lipids and without which many Ras-related proteins and nuclear lamins would not be prenylated and functional.
Assuntos
Genes ras , Neoplasias/genética , Neoplasias/terapia , Proteínas ras/metabolismo , Animais , Morte Celular , Humanos , Modelos Biológicos , Mutação , Neoplasias/patologia , Transdução de SinaisRESUMO
The receptor (R) for epidermal growth factor (EGF) is expressed at high levels on human breast cancer cells and associates with ErbB2, ErbB3, and Src proto-oncogene family protein tyrosine kinases (PTKs) to form membrane-associated PTK complexes with pivotal signaling functions. Recombinant human EGF was conjugated to the soybean-derived PTK inhibitor genistein (Gen) to construct an EGF-R-directed cytotoxic agent with PTK inhibitory activity. The EGF-Gen conjugate was capable of binding to and entering EGF-R-positive MDA-MB-231 and BT-20 breast cancer cells (but not EGF-R-negative NALM-6 or HL-60 leukemia cells) via its EGF moiety, and it effectively competed with unconjugated EGF for target EGF-R molecules in ligand binding assays. EGF-Gen inhibited the EGF-R tyrosine kinase in breast cancer cells at nanomolar concentrations, whereas the IC50 for unconjugated Gen was >10 microM. Notably, EGF-Gen triggered a rapid apoptotic cell death in MDA-MB-231 as well as BT-20 breast cancer cells at nanomolar concentrations. The EGF-Gen-induced apoptosis was EGF-R-specific because cells treated with the control granulocyte-colony stimulating factor-Gen conjugate did not become apoptotic. Apoptosis was dependent both on the PTK inhibitory function of Gen and the targeting function of EGF, because cells treated with unconjugated Gen plus unconjugated EGF did not undergo apoptosis. The IC50s of EGF-Gen versus unconjugated Gen against MDA-MB-231 and BT-20 cells in clonogenic assays were 30 +/- 3 nM versus 120 +/- 18 microM (P < 0.001) and 30 +/- 10 nM versus 112 +/- 17 microM (P < 0.001), respectively. Thus, the EGF-Gen conjugate is a >100-fold more potent inhibitor of EGF-R tyrosine kinase activity in intact breast cancer cells than unconjugated Gen and a >100-fold more potent cytotoxic agent against EGF-R+ human breast cancer cells than unconjugated Gen. Taken together, these results indicate that the EGF-R-associated PTK complexes have vital antiapoptotic functions in human breast cancer cells and may therefore be used as therapeutic targets.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/terapia , Fator de Crescimento Epidérmico/farmacologia , Genisteína/farmacologia , Proteínas Tirosina Quinases/efeitos dos fármacos , Idoso , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fragmentação do DNA/efeitos dos fármacos , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Genisteína/química , Genisteína/metabolismo , Humanos , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Contemporary therapies for acute myeloid leukemia (AML) commonly fail to cure patients because of the emergence of drug resistance. Drug resistance in AML is multifactorial but can be associated with the overexpression of transmembrane transporter molecules, including P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP), or associated with inactivation of the p53 tumor suppressor gene, as well as overexpression of the anti-apoptotic protein bcl-2. We are investigating if novel recombinant biotherapeutics can circumvent these resistance mechanisms to effectively treat refractory AML. To target the lethal action of diphtheria toxin (DT) to high affinity granulocyte-macrophage colony-stimulating factor (GMCSF) receptors on AML blasts, we have produced a recombinant chimeric fusion toxin, DTctGMCSF. Since DTctGMCSF enters and kills its target cells by unique mechanisms (GMCSF-receptor binding and protein synthesis inhibition) and is not similar in structure to Pgp or MRP substrates, we postulated that it would be an active agent against therapy-resistant AML. DTctGMCSF was selectively cytotoxic (IC50 1-10ng/ml) to GMCSF-receptor positive AML cells expressing the Pgp- or MRP-associated multi-drug resistant phenotypes, despite high level resistance to conventional chemotherapeutic agents. DTctGMCSF also efficiently killed AML cells deficient in p53 expression, as well as radiation-resistant AML cells and mixed lineage leukemia cells expressing high levels of bcl-2. In addition, DTctGMCSF killed > 99% of primary leukemic progenitor cells from therapy-refractory AML patients under conditions that we have previously found to not adversely affect the proliferative capacity or differentiation of pluripotent normal hematopoietic progenitor cells. DTctGMCSF may prove useful in treating myeloid leukemias that are otherwise resistant to a wide range of conventional therapies.
Assuntos
Toxina Diftérica/farmacologia , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/radioterapia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Antineoplásicos/farmacologia , Toxina Diftérica/genética , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mieloide/patologia , Tolerância a Radiação , Proteínas Recombinantes de Fusão/genética , Células-Tronco/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Human granulocyte-macrophage colony stimulating factor (GMCSF) and its high affinity receptor function to regulate the proliferation and differentiation of myeloid lineage hematopoietic cells, and may participate in the pathogenesis of many malignant myeloid diseases. We have used genetic engineering based on the elucidated molecular structures of human granulocyte-macrophage colony-stimulating factor and diphtheria toxin (DT) to produce a recombinant fusion toxin, DTctGMCSF, that targets diphtheria toxin to high affinity GMCSF receptors expressed on the surface of blast cells from a large fraction of patients with acute myeloid leukemia (AML). DTctGMCSF was specifically immunoreactive with antidiphtheria toxin and anti-GMCSF antiseras, and exhibited the characteristic catalytic activity of diphtheria toxin, catalyzing the in vitro ADP-ribosylation of purified elongation factor 2. The cytotoxic effects of DTctGMCSF were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium (MTT) bromide assay of cell viability and in vivo assays of protein synthesis inhibition. DTctGMCSF were specifically cytotoxic to human leukemia cell lines bearing high affinity receptors for human GMCSF with IC50 of 10(-9) to 10(-11) M. It was not toxic to mammalian hematopoietic cell lines lacking human GMCSF (hGMCSF) receptors. In receptor positive cells, cytotoxicity can be specifically blocked by a large excess of hGMCSF, confirming that its cytotoxicity is mediated through the hGMCSF receptor. THough DTctGMCSF inhibited granulocyte-macrophage colony formation by committed myeloid progenitor cells (CFU-GM), it did not significantly affect erythroid burst formation by committed erythroid progenitor cells (BFU-E), or mixed granulocyte-erythroid-macrophage-megakaryocyte colony formation by pluripotent multilineage progenitor cells (CFU-GEMM). DTctGMCSF holds promise for the treatment of myeloid lineage malignancies, and is a useful reagent to study hematopoiesis.
Assuntos
Toxina Diftérica/farmacologia , Leucemia/tratamento farmacológico , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antineoplásicos/farmacologia , Medula Óssea/efeitos dos fármacos , Células da Medula Óssea , Divisão Celular/efeitos dos fármacos , Toxina Diftérica/genética , Toxina Diftérica/imunologia , Ensaios de Seleção de Medicamentos Antitumorais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Soros Imunes , Leucemia/genética , Leucemia/patologia , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologia , Camundongos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas Recombinantes de Fusão/genética , Células-Tronco/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
We examined the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF), interleukin 3 (rhIL-3) and interleukin 6 (rhIL-6) on the radiation sensitivity of normal and leukemic bone marrow progenitor cell populations. Conditioning of leukemic progenitor cells (LPC) from acute lymphoblastic leukemia (ALL) patients with rhG-CSF enhanced their radiation sensitivity, whereas conditioning with rhIL-3 or rhIL-6 had the opposite effect. In contrast to its effects on LPC derived from ALL patients, rhG-CSF reduced the radiation sensitivity of normal myeloid progenitor cells as well as LPC from acute myeloblastic leukemia (AML) patients. Differential modulation of the radiation sensitivity of LPC by rhG-CSF may provide the basis for better total body irradiation (TBI) regimens for ALL patients undergoing autologous bone marrow transplantation (BMT).
Assuntos
Medula Óssea/efeitos da radiação , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Células-Tronco Hematopoéticas/efeitos da radiação , Leucemia-Linfoma Linfoblástico de Células Precursoras/radioterapia , Medula Óssea/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Terapia Combinada , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/radioterapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Protetores contra Radiação/farmacologia , Proteínas Recombinantes/uso terapêutico , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
Multiagent chemotherapy regimens fail to cure more than one-half of the patients with acute myeloid leukemia (AML) because of the emergence of dominant multidrug-resistant subclones of leukemia cells. We have developed a recombinant diphtheria toxin-human granulocyte macrophage colony-stimulating factor chimeric fusion protein (DTctGMCSF) that specifically targets GMCSF receptor-positive AML cells. This novel biotherapeutic agent induced rapid apoptotic cell death of chemotherapy-resistant AML cell lines and primary leukemic cells from treatment-refractory AML patients. Our results suggest that DTctGMCSF may be useful in the treatment of AML patients whose leukemia has recurred and developed resistance to contemporary chemotherapy programs.
Assuntos
Apoptose/fisiologia , Toxina Diftérica/toxicidade , Resistência a Múltiplos Medicamentos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/toxicidade , Imunotoxinas/toxicidade , Tolerância a Radiação , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Proteínas Recombinantes de Fusão/toxicidade , Doença Aguda , Apoptose/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia Mieloide , Leucemia Mieloide Aguda , Células Tumorais CultivadasRESUMO
Bruton's tyrosine kinase (BTK) is a member of the SRC-related TEC family of protein tyrosine kinases (PTKs). DT-40 lymphoma B cells, rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout, did not undergo radiation-induced apoptosis, but cells with disrupted lyn or syk genes did. Introduction of the wild-type, or a SRC homology 2 domain or a plecstrin homology domain mutant (but not a kinase domain mutant), human btk gene into BTK-deficient cells restored the apoptotic response to radiation. Thus, BTK is the PTK responsible for triggering radiation-induced apoptosis of lymphoma B cells, and its kinase domain is indispensable for the apoptotic response.
Assuntos
Apoptose , Linfócitos B/efeitos da radiação , Linfoma de Células B/patologia , Proteínas Tirosina Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/citologia , Linfócitos B/enzimologia , Galinhas , Raios gama , Marcação de Genes , Humanos , Imunoglobulina M/imunologia , Linfoma de Células B/enzimologia , Fosforilação , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/fisiologia , Células Tumorais Cultivadas , Domínios de Homologia de srcRESUMO
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.
Assuntos
Linfócitos B/enzimologia , Proteína Quinase CDC2/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/efeitos da radiação , Sítios de Ligação/genética , Proteína Quinase CDC2/química , Proteína Quinase CDC2/genética , Reparo do DNA , Fase G2/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tirosina/química , Tirosina/efeitos da radiação , Quinases da Família src/química , Quinases da Família src/genéticaRESUMO
Here we provide experimental evidence that ionizing radiation induces inhibitory tyrosine phosphorylation of the p34cdc2 kinase in human leukemic B-cell precursors. Herbimycin A markedly reduced tyrosine phosphorylation of p34cdc2 in irradiated leukemic B-cell precursors, thereby preventing radiation-induced cell cycle arrest at the G2-M transition checkpoint. Thus, tyrosine phosphorylation is directly responsible for the inactivation of p34cdc2 in irradiated human leukemic B-cell precursors and activation of protein tyrosine kinases is a proximal and mandatory step in radiation-induced G2-arrest arrest at the G2-M checkpoint. Human WEE1 kinase isolated from unirradiated or irradiated leukemic B-cell precursors had minimal tyrosine kinase activity towards p34cdc2. We detected no increase of human WEE1 kinase activity after radiation of leukemic B-cell precursors, as measured by (a) autophosphorylation, (b) tyrosine phosphorylation of a synthetic peptide derived from the p34cdc2 amino-terminal region or (c) recombinant human p34cdc2-cyclin B complex. Thus the signaling pathway leading to inhibitory tyrosine phosphorylation of p34cdc2 and G2-arrest in irradiated human leukemic B-cell precursors functions independent of p49 WEE1 HU and enzymes which augment the tyrosine kinase activity of p49 WEE 1HU.
Assuntos
Linfócitos B/efeitos da radiação , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular , Fase G2/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos da radiação , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/efeitos da radiação , Proteínas Nucleares , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Processamento de Proteína Pós-Traducional/efeitos da radiação , Linfócitos B/metabolismo , Linfócitos B/patologia , Benzoquinonas , Proteína Quinase CDC2/antagonistas & inibidores , Ativação Enzimática/efeitos da radiação , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Lactamas Macrocíclicas , Substâncias Macromoleculares , Fator Promotor de Maturação/fisiologia , Proteínas de Neoplasias/antagonistas & inibidores , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos da radiação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Proteínas Tirosina Quinases/fisiologia , Quinonas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Rifabutina/análogos & derivados , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
B-cell precursor (BCP) leukemia is the most common form of childhood cancer and represents one of the most radiation-resistant forms of human malignancy. In this study, we examined the antileukemic efficacy of the B43 (anti-CD19)-pokeweed antiviral protein (B43-PAP) immunotoxin against radiation-resistant BCP leukemia cells. B43-PAP caused apoptosis of radiation-resistant primary BCP leukemia cells, killed greater than 99% of radiation-resistant primary leukemic progenitor cells from BCP leukemia patients, and conferred extended survival to severe combined immunodeficiency (SCID) mice xenografted with radiation-resistant human BCP leukemia. Furthermore, the combination of B43-PAP and total body irradiation (TBI) was more effective than TBI alone in two SCID mouse bone marrow transplantation models of radiation-resistant human BCP leukemia. Thus, B43-PAP may prove useful in the treatment of radiation-resistant BCP leukemia.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antivirais/farmacologia , Linfoma de Burkitt/tratamento farmacológico , Imunotoxinas/farmacologia , N-Glicosil Hidrolases , Proteínas de Plantas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Transplante de Medula Óssea , Linfoma de Burkitt/radioterapia , Linfoma de Burkitt/cirurgia , Criança , Terapia Combinada , Humanos , Técnicas In Vitro , Camundongos , Camundongos SCID , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos da radiação , Tolerância a Radiação , Proteínas Inativadoras de Ribossomos Tipo 1 , Transplante Heterólogo , Transplante Isogênico , Irradiação Corporal TotalRESUMO
CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.
Assuntos
Antígenos CD19/metabolismo , Apoptose , Linfoma de Burkitt/metabolismo , Quinases da Família src/metabolismo , Animais , Antígenos CD19/imunologia , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Inibidores Enzimáticos/uso terapêutico , Genisteína , Humanos , Imunoconjugados/uso terapêutico , Imunotoxinas/uso terapêutico , Isoflavonas/uso terapêutico , Camundongos , Camundongos SCID , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína Supressora de Tumor p53RESUMO
The ability of total body irradiation (TBI) to eradicate clonogenic leukemia cells from B-lineage acute lymphoblastic leukemia patients prior to bone marrow transplantation (BMT) is greatly hampered by their inherent or acquired radiation resistance. The radiorefractory nature of these cells is believed to contribute to the high relapse rate subsequent to TBI and BMT in patients with B-lineage acute lymphoblastic leukemia (ALL). A method by which clonogenic leukemia cells could be radiosensitized in vivo could be clinically beneficial. In the present study, we used a highly radiation resistant subclone of the murine B-lineage leukemia cell line BCL-1 in a syngeneic BMT model to investigate if any of the B-cell stimulatory cytokines interleukin 2, interleukin 4, interleukin 5, or interleukin 6 could have radiosensitizing effects. All untreated BALB/c mice (N = 33) inoculated with 1 x 10(6) BCL-1 cells died of disseminated leukemia within 24 days with a median survival of 13.3 days. TBI (700 cGy = LD100/30 for BALB/c mice) followed by syngeneic BMT (N = 70) extended the median survival to 23.6 days (P < 0.001 by log-rank test). A single intraperitoneal bolus injection of 100 ng, 500 ng, or 2500 ng recombinant murine interleukin 6(rmIL-6) 2-4 hours before TBI extended the median survival to 32.5 days, 31.0 days, and 30.5 days, respectively (P < 0.01 by log-rank test for all dose groups). The improved survival was not due to any direct anti-leukemic activity of rmIL-6 and all control BALB/c mice (N = 15) that received the same doses of rmIL-6 but did not undergo TBI and BMT died of BCL-1 leukemia within 28 days with a median survival of 13.6 days. In contrast to rmIL-6, recombinant murine interleukin 5 (rmIL-5) had minimal radiosensitizing effects.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Apoptose , Transplante de Medula Óssea , Interleucina-6/farmacologia , Leucemia de Células B/terapia , Leucemia Experimental/terapia , Radiossensibilizantes/farmacologia , Animais , Feminino , Humanos , Leucemia de Células B/patologia , Leucemia de Células B/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Transplante Isogênico , Irradiação Corporal TotalRESUMO
Topotecan [(S)-9-dimethylaminomethyl-10-hydroxycamptothecin hydrochloride; SK&F 104864-A, NSC 609699], a water soluble semisynthetic analogue of the alkaloid camptothecin, is a potent topoisomerase I inhibitor. Here we show that topotecan stabilizes topoisomerase I/DNA cleavable complexes in radiation-resistant human B-lineage acute lymphoblastic leukemia (ALL) cells, causes rapid apoptotic cell death despite high-level expression of bcl-2 protein, and inhibits ALL cell in vitro clonogenic growth in a dose-dependent fashion. Furthermore, topotecan elicited potent antileukemic activity in three different severe combined immunodeficiency (SCID) mouse models of human poor prognosis ALL and markedly improved event-free survival of SCID mice challenged with otherwise fatal doses of human leukemia cells at systemic drug exposure levels that can be easily achieved in children with leukemia.
Assuntos
Linfoma de Burkitt/tratamento farmacológico , Camptotecina/análogos & derivados , Adolescente , Adulto , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Apoptose , Camptotecina/farmacocinética , Camptotecina/uso terapêutico , Criança , Pré-Escolar , Dano ao DNA , Feminino , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos SCID , Transplante de Neoplasias , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Análise de Sobrevida , Inibidores da Topoisomerase I , TopotecanRESUMO
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.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Imunoconjugados/uso terapêutico , Isoflavonas/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Anticorpos Monoclonais , Antígenos CD19 , Apoptose , Dano ao DNA , DNA de Neoplasias/metabolismo , Genisteína , Imunoconjugados/administração & dosagem , Imunoconjugados/farmacocinética , Isoflavonas/administração & dosagem , Isoflavonas/farmacocinética , Infiltração Leucêmica , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Camundongos , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
As presently reported, both ionizing radiation and engagement of the CD19 receptor are capable of inducing apoptosis in B-lineage acute lymphoblastic leukemia (ALL) cells. In both instances, activation of tyrosine kinases appears to be a proximal and mandatory step, since it can be prevented by the tyrosine kinase inhibitor genistein. This common biochemical signaling pathway involves the rapid activation of the Src family tyrosine kinase LCK (p56lck), which is physically associated with the CD19 receptor, and enhanced tyrosine phosphorylation of multiple substrates leading to stimulation of phosphoinositide turnover, and activation of protein kinase C. Importantly, engagement of the CD19 receptor promoted radiation-induced apoptosis in radiation-resistant B-lineage ALL cells in a cell type-specific fashion. Our results prompt the hypothesis that clonogenic B-lineage ALL blasts with an inherent or acquired resistance to radiation could be radiosensitized in clinical settings using anti-CD19 MoAb B43 or its homoconjugate as adjuncts.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Apoptose/efeitos da radiação , Linfoma de Burkitt/patologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Antígenos CD19 , Linfoma de Burkitt/enzimologia , Ativação Enzimática , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Tolerância a Radiação , Células Tumorais CultivadasRESUMO
We examined bone marrow and peripheral blood specimens from pediatric acute lymphoblastic leukemia (ALL) patients after autologous bone marrow transplantation (BMT) as well as fetal lymphohematopoietic tissues for the presence of CD5+ B lymphocytes. CD5+ B lymphocytes represented 23.6 +/- 0.7% of CD19+ fetal spleen B lineage lymphoid cells, 24.2 +/- 2.5% of CD19+ fetal bone marrow B lineage lymphoid cells and 18.1 +/- 1.7% of CD19+ fetal liver B lineage lymphoid cells. By comparison, in normal pediatric bone marrow samples, only 1.5 +/- 0.3% of lymphoid cells and 4.3 +/- 0.2% of CD19+ B lineage lymphoid cells expressed CD5 antigen. Similarly, very few CD5+CD19+ cells (< or = 2% of lymphoid cells) were found in day 30 and day 100 post-BMT bone marrow or peripheral blood specimens from B lineage ALL patients undergoing autologous BMT using autografts purged ex vivo with B43(anti-CD19)-PAP immunotoxin plus 4-hydroperoxycyclophosphamide (4-HC). Two bone marrow samples that were obtained and analyzed 1 year post-BMT contained only 2 to 4% CD5+CD19+ cells, accounting for 5 to 7% of the total CD19+ population. The fraction of CD5+ B lymphocytes in post-BMT bone marrow samples was not greater than the fraction of CD5+ B lymphocytes in normal healthy bone marrow.
Assuntos
Antígenos CD/análise , Linfócitos B/citologia , Linfócitos B/imunologia , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/patologia , Desenvolvimento Embrionário e Fetal/imunologia , Anticorpos Monoclonais , Antígenos CD19 , Antígenos de Diferenciação de Linfócitos B/análise , Linfócitos B/fisiologia , Medula Óssea/embriologia , Medula Óssea/imunologia , Células da Medula Óssea , Antígenos CD5 , Diferenciação Celular/fisiologia , Feto/citologia , Feto/imunologia , Citometria de Fluxo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/cirurgia , Baço/citologia , Baço/embriologia , Baço/imunologia , Transplante AutólogoRESUMO
The radiation sensitivity of primary clonogenic blasts from 44 children with newly diagnosed B-cell precursor acute lymphoblastic leukemia (ALL) was analyzed using leukemic progenitor cell (LPC) colony assays. The derived values for SF2 (surviving fraction at 200 cGy) and alpha (initial slope of radiation survival curves constructed according to the linear quadratic model) indicated a marked interpatient heterogeneity in intrinsic radiation sensitivity of LPC populations. The SF2 values ranged from 0.01 to 1.00 (median = 0.430; mean +/- SE = 0.47 +/- 0.04), and the alpha values ranged from 0.000 to 3.272 Gy-1 (median = 0.280 Gy-1; mean +/- SE = 0.430 +/- 0.093 Gy-1). When CD19+ CD34+ versus CD19+ CD34- immunophenotypes were compared, a trend toward higher SF2 and lower alpha values were observed in LPC from CD34+ patients, consistent with greater radiation resistance. When patients were divided into three approximately equal groups based on increasing levels of CD34 expression, a clear ordering effect was observed indicating that increased CD34 expression levels are associated with significantly higher radiation resistance at the level of B-lineage LPC. The highest CD34 expression group (> or = 75% positivity) had 1.4-fold higher SF2 (P = 0.05) and twofold lower alpha values (P = 0.06) than the lowest group (< 30% positivity). Furthermore, the CD34 positivity of radiation resistant (alpha < or = 0.2 and SF2 > or = 0.5) B-cell precursor ALL cases was greater than the CD34 positivity of radiation sensitive (alpha > 0.2 and/or SF2 < 0.5) cases (56 +/- 9% versus 34 +/- 9%, P = 0.09). Whereas only 6 of 16 (38%) of radiation sensitive cases were CD34+, 11 of 15 (73%) of radiation resistant cases expressed CD34 (P = 0.04). Our results offer new insights into the inherent and/or acquired radiation resistance of primary clonogenic blasts from B-cell precursor ALL patients.
Assuntos
Linfócitos B/efeitos da radiação , Crise Blástica/patologia , Medula Óssea/efeitos da radiação , Linfoma de Burkitt/patologia , Células-Tronco Hematopoéticas/efeitos da radiação , Fatores Etários , Antígenos CD/análise , Linfócitos B/imunologia , Linfócitos B/patologia , Crise Blástica/imunologia , Medula Óssea/patologia , Linfoma de Burkitt/imunologia , Criança , Pré-Escolar , Feminino , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Humanos , Imunofenotipagem , Masculino , Ensaio Tumoral de Célula-TroncoRESUMO
The radiobiologic features of primary clonogenic blasts (referred to also as T-lineage leukemic progenitor cells) from newly diagnosed and relapsed T-lineage acute lymphoblastic leukemia (ALL) patients were analyzed. Intrinsic radiation sensitivity differed substantially among primary clonogenic blasts from 34 newly diagnosed patients. The mean D0 (37% dose slope), SF2 (surviving fraction at 200 cGy), and alpha values (initial slope of the survival curve) were 141 +/- 15 cGy, 0.31 +/- 0.04, and 0.630 +/- 0.093 Gy-1, respectively. Among newly diagnosed cases, nine had SF2 values of greater than or equal to 0.50 and alpha values of less than or equal to 0.2 Gy-1, consistent with a marked intrinsic radiation resistance at the level of clonogenic blasts using the multitarget and linear quadratic models of cell survival. Of these nine radiation resistant cases, seven were CD3+. Furthermore, the mean D0 (162 +/- 20.8 cGy) and SF2 (0.377 +/- 0.057) values for the 20 CD3+ cases were significantly higher than the D0 (108.6 +/- 18.2 cGy) and SF2 (0.204 +/- 0.051) values for the 14 CD3- cases (P less than or equal to .05). Thus, clonogenic blasts from CD3+ newly diagnosed T-lineage ALL patients were more resistant to radiation than clonogenic blasts from CD3- newly diagnosed T-lineage ALL patients. Nineteen T-lineage ALL patients received autologous bone marrow transplants during complete remission. Pretransplant conditioning consisted of total body irradiation (TBI) combined with high-dose chemotherapy. Primary clonogenic blasts from patients who relapsed after bone marrow transplantation (BMT) displayed a particularly high degree of intrinsic radiation resistance with a mean D0 value of 333 cGy and an alpha value of 0.112 Gy-1. The expression of CD3 antigen appeared to predict the outcome of relapsed T-lineage ALL patients undergoing autologous BMT after TBI plus high-dose chemotherapy. The Kaplan-Meier estimates and standard errors of the probability of remaining in remission after BMT were 60% +/- 22% (mean relapse - free interval = 1.6 +/- 0.7 years) for CD3- patients and 0% +/- 0% (mean relapse - free interval = 0.2 +/- 0.0 years) for CD3+ patients (P = .002). Furthermore, the mean percentage of CD3-positive leukemic marrow blasts at presentation or relapse before BMT was significantly lower than the mean percentage of CD3-positive leukemic marrow blasts at relapse after BMT. Notably, in cultured leukemic bone marrow specimens from newly diagnosed as well as relapsed patients, colony blasts surviving in vitro radiation expressed CD3 more vividly than did colony blasts in unirradiated cultures.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos de Diferenciação de Linfócitos T/análise , Leucemia-Linfoma de Células T do Adulto/radioterapia , Receptores de Antígenos de Linfócitos T/análise , Adolescente , Adulto , Antígenos CD/análise , Transplante de Medula Óssea/patologia , Complexo CD3 , Ciclo Celular , Sobrevivência Celular/efeitos da radiação , Criança , Pré-Escolar , Células Clonais/efeitos da radiação , Feminino , Humanos , Técnicas In Vitro , Leucemia-Linfoma de Células T do Adulto/imunologia , Masculino , Análise de Sobrevida , Subpopulações de Linfócitos T/fisiologia , Irradiação Corporal TotalRESUMO
The purposes of this study were to examine the biologic effects of the engagement of the interleukin-7 receptor (IL-7R) with recombinant human interleukin-7 (rhIL-7) in immunophenotypically distinct T-lineage acute lymphoblastic leukemia (ALL) blasts and to elucidate the biochemical nature of the IL-7R-linked transmembrane signal in rhIL-7-responsive T-lineage ALL blast populations. In the absence of costimulants, rhIL-7 stimulated the in vitro proliferation and colony formation of freshly isolated leukemic blasts from six to eight T-lineage ALL patients with a mean plating efficiency of 196 +/- 53 (background subtracted) colonies/10(5) blasts plated. Stimulation of T-lineage ALL blasts with rhIL-7 resulted in markedly enhanced tyrosine phosphorylation of six distinct phosphoproteins with molecular weights of 57, 72, 98, 123, 150, and 190 Kd, and induced a rapid increase in the production of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3), which was inhibitable by the tyrosine-specific protein kinase inhibitor genistein, but not by the serine/threonine-specific protein kinase C inhibitor H7. Similarly, rhIL-7 stimulated Ins-1,4,5-P3 production in CEM-1.3 T-lineage ALL cells and this stimulation was inhibitable by the tyrosine-specific protein kinase inhibitors genistein and herbimycin A, but not by H-7. Thus, the transmembrane signal triggered by engagement of the IL-7R is intimately linked to a functional tyrosine-specific protein kinase pathway and stimulates the phosphoinositide (PI) turnover and proliferation of T-lineage ALL blasts. The presented data confirm and extend previous studies on the expression of functional IL-7R on T-lineage ALL blasts and support the hypothesis that IL-7 may play an important regulatory role in the biology of T-lineage ALL.