RESUMEN
The composite non-nucleoside reverse transcriptase inhibitor (NNRTI) binding pocket model was used to study a number of thiourea analogues with different substitutions at the 4-phenyl position including N-[2-(4-methylphenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea (compound HI-244), which inhibited recombinant RT better than trovirdine or compound HI-275 with an unsubstituted phenyl ring. HI-244 effectively inhibited the replication of HIV-1 strain HTLV(IIIB) in human peripheral blood mononuclear cells with an IC50 value of 0.007 microM, which is equal to the IC50 value of trovirdine. Notably, HI-244 was 20 times more effective than trovirdine against the multidrug-resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74V, 41L and 215Y) and seven times more potent than trovirdine against the NNRTI-resistant HIV-1 strain A17 with a Y181C mutation.
Asunto(s)
Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Resistencia a Múltiples Medicamentos , VIH-1/efectos de los fármacos , Piridinas/química , Piridinas/farmacología , ADN Polimerasa Dirigida por ARN/metabolismo , Tiourea/análogos & derivados , Sitios de Unión , VIH-1/fisiología , Humanos , Concentración 50 Inhibidora , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Modelos Moleculares , Conformación Proteica , Piridinas/metabolismo , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/metabolismo , Relación Estructura-Actividad , Tiourea/química , Tiourea/metabolismo , Tiourea/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
We have replaced the pyridyl ring of trovirdine with an alicyclic cyclohexenyl, adamantyl or cis-myrtanyl ring. Only the cyclohexenyl-containing thiourea compound N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]- thiourea (HI-346) (as well as its chlorine-substituted derivative N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]- thiourea/HI-445) showed RT inhibitory activity. HI-346 and HI-445 effectively inhibited recombinant RT with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cell-free RT inhibition assays was: HI-346 (IC50 = 0.4 microM) > HI-445 (IC50 = 0.5 microM) > trovirdine (IC50 = 0.8 microM) > MKC-442 (IC5 = 0.8 microM) = delavirdine (IC50 = 1.5 microM) > nevirapine (IC50 = 23 microM). In accord with this data, both compounds inhibited the replication of the drug-sensitive HIV-1 strain HTLV(IIIB) with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cellular HIV-1 inhibition assays was: HI-445 = HI-346 (IC50 = 3 nM) > MKC-442 (IC50 = 4 nM) = AZT (IC50 = 4 nM) > trovirdine (IC50 = 7 nM) > delavirdine (IC50 = 9 nM) > nevirapine (IC50 = 34 nM). Surprisingly, the lead compounds HI-346 and HI-445 were 3-times more effective against the multidrug resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74V,41L, and 215Y) than they were against HTLV(IIIB) with wild-type RT. HI-346 and HI-445 were 20-times more potent than trovirdine, 200-times more potent than AZT, 300-times more potent than MKC-442, 400-times more potent than delavirdine, and 5000-times more potent than nevirapine against the multidrug resistant HIV-1 strain RT-MDR. HI-445 was also tested against the RT Y181C mutant A17 strain of HIV-1 and found to be >7-fold more effective than trovirdine and >1,400-fold more effective than nevirapine or delavirdine. Similarly, both HI-346 and HI-445 were more effective than trovirdine, nevirapine, and delavirdine against the problematic NNI-resistant HIV-1 strain A17-variant with both Y181C and K103N mutations in RT, although their activity was markedly reduced against this strain. Neither compound exhibited significant cytotoxicity at effective concentrations (CC50 >100 microM). These findings establish the lead compounds HI-346 and HI-445 as potent inhibitors of drug-sensitive as well as multidrug-resistant stains of HIV-1.
Asunto(s)
Aminopiridinas/farmacología , Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Tiourea/análogos & derivados , Aminopiridinas/química , Fármacos Anti-VIH/química , Farmacorresistencia Microbiana , Resistencia a Múltiples Medicamentos , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/efectos de los fármacos , Humanos , Modelos Moleculares , Inhibidores de la Transcriptasa Inversa/química , Tiourea/química , Tiourea/farmacologíaRESUMEN
A hospital-based facility for in vivo prompt gamma neutron activation analysis of nitrogen for body protein determination is described. The patient is laid on a movable couch and is scanned with a vertically collimated neutron beam from a 252Cf neutron source (the amount of Cf varying from 120 to 40 microg due to the physical decay) positioned below the patient. Four large NaI(Tl) detectors are used to measure the 10.8 MeV gamma-rays from nitrogen. To check the long-term stability of the system, a solid phantom simulating the geometry of the adult human trunk, having similar elemental composition as tissue, was constructed. Repeated phantom measurements over 6 months gave a reproducibility in nitrogen determination of 2.9% (1 SD). Duplicate patient measurements carried out within a week showed a reproducibility of 5% (1 SD). A calibration method for absolute protein measurements in patients is presented. Patients are normally measured for 40 min; giving a mean whole-body equivalent dose of 0.25 mSv. Results from measurements on 13 cancer patients are presented.
Asunto(s)
Monitoreo Fisiológico/instrumentación , Proteínas de Neoplasias/análisis , Adulto , Anciano , Calibración , Californio , Electrónica Médica , Femenino , Rayos gamma , Humanos , Masculino , Persona de Mediana Edad , Neutrones , Nitrógeno/análisis , Fantasmas de Imagen , Sensibilidad y EspecificidadRESUMEN
The thiophene-ethyl thiourea (TET) compound N'-[2-(2-thiophene)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea (compound HI-443) was five times more potent than trovirdine, 1250 times more potent than nevirapine, 100 times more potent than delavirdine, 75 times more potent than MKC-442, and 50 times more potent than AZT against the multidrug resistant HIV-1 strain RT-MDR with a V106A mutation. HI-443 was almost as potent against the NNI-resistant HIV-1 strain A17 with a Y181C mutation as it was against HTLV(IIIB). The activity of HI-443 against A17 was ten times more potent than that of trovirdine, 2083 times more potent than that of nevirapine, and 1042 times more potent than that of delavirdine. HI-443 inhibited the replication of the NNI-resistant HIV-1 strain A17 variant with Y181C plus K103N mutations in RT with an IC50 value of 3.3 microM, whereas the IC50 values of trovirdine, nevirapine, and delavirdine were all >100 microM. These findings establish the novel thiophene containing thiourea compound HI-443 as a novel NNI with potent antiviral activity against NNI-sensitive, NNI-resistant and multidrug-resistant strains of HIV-1.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Piridinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Tiourea/análogos & derivados , Células Cultivadas , VIH-1/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Piridinas/química , Tiourea/química , Tiourea/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
A series of novel phenethylthiazolylthiourea (PETT) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of HIV reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket. The structure-based design and synthesis of these new PETT derivatives were complemented by biological assays of their anti-HIV activity. Modeling studies for rational drug design included the construction of a composite NNI binding pocket from nine RT-NNI crystal structures, the analyses of surface complementarity between NNI and RT, and application of Ki calculations combined with a docking procedure involving the novel PETT derivatives. The use of the composite NNI binding pocket allowed the identification and structure-based design of three promising PETT derivatives with ortho-F (2), ortho-Cl (3), and meta-F (5) substituents on the phenyl ring. These novel PETT derivatives were more active than AZT or trovirdine and showed potent anti-HIV activity with IC50[p24] values of < 1 nM and selectivity indices of > 100,000.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Sitios de Unión , Cloro/química , Diseño de Fármacos , Flúor/química , Transcriptasa Inversa del VIH/metabolismo , Humanos , Modelos Moleculares , Inhibidores de la Transcriptasa Inversa/metabolismo , Relación Estructura-Actividad , TioureaRESUMEN
Two highly potent dihydroalkoxybenzyloxopyrimidine (DABO) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket and tested for anti-HIV activity. Our lead DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-on e, elicited potent inhibitory activity against purified recombinant HIV RT and abrogated HIV replication in peripheral blood mononuclear cells at nanomolar concentrations (50% inhibitory concentration, <1 nM) but showed no detectable cytotoxicity at concentrations as high as 100 microM.
Asunto(s)
Transcriptasa Inversa del VIH/metabolismo , Pirimidinonas/síntesis química , Inhibidores de la Transcriptasa Inversa/síntesis química , Sulfuros/síntesis química , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Transcriptasa Inversa del VIH/química , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Pirimidinonas/química , Pirimidinonas/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Sulfuros/química , Sulfuros/farmacologíaRESUMEN
Leukemic cells from bone marrow (BM) of 17 infants and 127 children with newly diagnosed ALL, as well as fetal liver and BM and normal infant BM samples, were analyzed for presence of a t(4;11) translocation using standard cytogenetic techniques and expression of an MLL-AF4 fusion transcript using standard reverse transcriptase-polymerase chain reaction (RT-PCR) assays as well as nested RT-PCR that is 100-fold more sensitive than standard RT-PCR. Overall, 9 of 17 infants and 17 of 127 noninfant pediatric ALL patients were positive for expression of MLL-AF4 fusion transcripts, as determined by standard and/or nested RT-PCR assays. None of the MLL-AF4(+) cases were positive for E2A-PBX1 or BCR-ABL fusion transcript expression. Although 8 of 9 MLL-AF4(+) infants had cytogenetically detectable t(4;11)(q21;q23), 15 of the 17 MLL-AF4(+) noninfants were t(4;11)-. Infants with MLL-AF4(+) ALL had poor outcomes, whereas non-infant MLL-AF4(+)/t(4;11)- patients had favorable outcomes similar to MLL-AF4(-) patients. Notably, MLL-AF4 transcripts also were detected by nested RT-PCR in 4 of 16 fetal BMs, 5 of 13 fetal livers, and 1 of 6 normal infant BMs, but not in any of the 44 remission BM specimens from pediatric ALL patients. Our results provide unprecedented evidence that MLL-AF4 fusion transcripts can be present in normal hematopoietic cells, indicating that their expression is insufficient for leukemic transformation of normal lymphocyte precursors.
Asunto(s)
Biomarcadores de Tumor/análisis , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 4/genética , Células Madre Hematopoyéticas/metabolismo , Proteínas de Neoplasias/análisis , Células Madre Neoplásicas/metabolismo , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocación Genética/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Médula Ósea/patología , Trasplante de Médula Ósea , Transformación Celular Neoplásica/genética , Niño , Preescolar , Cromosomas Humanos Par 11/ultraestructura , Cromosomas Humanos Par 4/ultraestructura , Terapia Combinada , Supervivencia sin Enfermedad , Femenino , Proteínas Fetales/análisis , Regulación del Desarrollo de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Lactante , Hígado/embriología , Hígado/patología , Masculino , Proteína de la Leucemia Mieloide-Linfoide , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/biosíntesis , Reacción en Cadena de la Polimerasa/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Pronóstico , Inducción de Remisión , Sensibilidad y Especificidad , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Here we present evidence that exposure of DT40 lymphoma B-cells to low energy electromagnetic fields (EMF) results in activation of phospholipase C-gamma 2 (PLC-gamma2), leading to increased inositol phospholipid turnover. PLC-gamma2 activation in EMF-stimulated cells is mediated by stimulation of the Bruton's tyrosine kinase (BTK), a member of the Src-related TEC family of protein tyrosine kinases, which acts downstream of LYN kinase and upstream of PLC-gamma2. B-cells rendered BTK-deficient by targeted disruption of the btk gene did not show enhanced PLC-gamma2 activation in response to EMF exposure. Introduction of the wild-type (but not a kinase domain mutant) human btk gene into BTK-deficient B-cells restored their EMF responsiveness. Thus, BTK exerts a pivotal and mandatory function in initiation of EMF-induced signaling cascades in B-cells.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Agammaglobulinemia Tirosina Quinasa , Animales , Pollos , Campos Electromagnéticos , Activación Enzimática , Isoenzimas/metabolismo , Linfoma de Células B/enzimología , Linfoma de Células B/patología , Fosfolipasa C gamma , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/metabolismoAsunto(s)
Composición Corporal/fisiología , Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/uso terapéutico , Proteínas/análisis , Absorciometría de Fotón/métodos , Adulto , Composición Corporal/efectos de los fármacos , Índice de Masa Corporal , Femenino , Rayos gamma , Humanos , Masculino , Persona de Mediana Edad , Nitrógeno/análisis , Isótopos de Nitrógeno , PlacebosRESUMEN
We have evaluated the clinical potential of TXU (anti-CD7)-pokeweed antiviral protein (PAP) immunoconjugate (TXU-PAP) as a new biotherapeutic anti-human immunodeficiency virus (anti-HIV) agent by evaluating its anti-HIV type 1 (anti-HIV-1) activity in vitro, as well as in a surrogate human peripheral blood lymphocyte-severe combined immunodeficient (Hu-PBL-SCID) mouse model of human AIDS. The present report documents in a side-by-side comparison the superior in vitro anti-HIV-1 activity of TXU-PAP compared to the activities of zidovudine, 2',3'-didehydro-2',3'-dideoxythymidine, unconjugated PAP, and B53-PAP, an anti-CD4-PAP immunoconjugate. Notably, TXU-PAP elicited potent anti-HIV activity in the Hu-PBL-SCID mouse model of human AIDS without any side effects and at doses that were very well tolerated by cynomolgus monkeys. Furthermore, plasma samples from TXU-PAP-treated cynomolgus monkeys showed potent anti-HIV-1 activity in vitro.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , VIH-1/efectos de los fármacos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , N-Glicosil Hidrolasas , Proteínas de Plantas/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/virología , Animales , Fármacos Anti-VIH/química , Modelos Animales de Enfermedad , Humanos , Macaca fascicularis , Ratones , Ratones SCID , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Estavudina/uso terapéutico , Zidovudina/uso terapéuticoRESUMEN
Here, we present evidence that exposure of DT40 lymphoma B cells to low energy electromagnetic field (EMF) results in a tyrosine kinase-dependent activation of phospholipase Cgamma2 (PLC-gamma2) leading to increased inositol phospholipid turnover. B cells rendered PLC-gamma2-deficient by targeted disruption of the PLC-gamma2 gene as well as PLC-gamma2-deficient cells reconstituted with Src homology domain 2 (SH2) domain mutant PLC-gamma2 did not show any increase in inositol-1,4,5-trisphosphate levels after EMF exposure, providing direct evidence that PLC-gamma2 is responsible for EMF-induced stimulation of inositol phospholipid turnover, and its SH2 domains are essential for this function. B cells rendered SYK-deficient by targeted disruption of the syk gene did not show PLC-gamma2 activation in response to EMF exposure. The C-terminal SH2 domain of SYK kinase is essential for its ability to activate PLC-gamma2. SYK-deficient cells reconstituted with a C-terminal SH2 domain mutant syk gene failed to elicit increased inositol phospholipid turnover after EMF exposure, whereas SYK-deficient cells reconstituted with an N-terminal SH2 domain mutant syk gene showed a normal EMF response. LYN kinase is essential for the initiation of this biochemical signaling cascade. Lymphoma B cells rendered LYN-deficient through targeted disruption of the lyn gene did not elicit enhanced inositol phospholipid turnover after EMF exposure. Introduction of the wild-type (but not a kinase domain mutant) mouse fyn gene into LYN-deficient B cells restored their EMF responsiveness. B cells reconstituted with a SH2 domain mutant fyn gene showed a normal EMF response, whereas no increase in inositol phospholipid turnover in response to EMF was noticed in LYN-deficient cells reconstituted with a SH3 domain mutant fyn gene. Taken together, these results indicate that EMF-induced PLC-gamma2 activation is mediated by LYN-regulated stimulation of SYK, which acts downstream of LYN kinase and upstream of PLC-gamma2.
Asunto(s)
Linfocitos B/enzimología , Campos Electromagnéticos , Precursores Enzimáticos/genética , Isoenzimas/metabolismo , Proteínas Tirosina Quinasas/genética , Fosfolipasas de Tipo C/metabolismo , Familia-src Quinasas/metabolismo , Animales , Pollos , Activación Enzimática/fisiología , Precursores Enzimáticos/deficiencia , Marcación de Gen , Inositol 1,4,5-Trifosfato/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Linfoma , Fosfatidilinositoles/metabolismo , Fosfolipasa C gamma , Proteínas Tirosina Quinasas/deficiencia , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fyn , Transducción de Señal/fisiología , Quinasa Syk , Dominios Homologos src/fisiologíaRESUMEN
Novel dihydroalkoxybenzyloxopyrimidine (S-DABO) derivatives targeting the non-nucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been synthesized using a novel computer model for the NNI binding pocket and tested for their RT inhibitory activity in cell-free assays using purified recombinant HIV RT as well as for their anti-HIV activity in HTL VIIIB-infected peripheral blood mononuclear cells. Our computational approach allowed the identification of several ligand derivatization sites for the generation of more potent S-DABO derivatives. Our lead S-DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one (compound 3), elicited potent anti-HIV activity with an IC50 value of less than 1nM for inhibition of HIV replication without any evidence of cytotoxicity and an unprecedented selectivity index of > 100,000.
Asunto(s)
Fármacos Anti-VIH/farmacología , Pirimidinonas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Sulfuros/farmacología , Fármacos Anti-VIH/química , Sistema Libre de Células , Diseño de Fármacos , VIH-1/efectos de los fármacos , Humanos , Monocitos/efectos de los fármacos , Monocitos/virología , Pirimidinonas/química , Inhibidores de la Transcriptasa Inversa/química , Sulfuros/químicaRESUMEN
We used reverse transcriptase polymerase chain reaction (RT-PCR) assays to examine primary leukemic cells in on-study diagnostic bone marrow specimens from 642 children with newly diagnosed acute lymphoblastic leukemia (ALL) for the expression of MLL-AF4, E2A-PBX1, and BCR-ABL fusion transcripts. All PCR assays were performed centrally in the Children's Cancer Group ALL Biology Reference Laboratory. MLL-AF4 transcript was found in only 0.7% of the study population which excluded infants. E2A-PBX1 transcript was found in 2.5% of the study population and 3.3% of B-precursor cases. Expression was associated with massive hepatomegaly. BCR-ABL transcript was found in 2.3% of cases and correlated with older age, induction failure, and inferior event-free survival (EFS). RT-PCR assays allow rapid identification of patients with MLL-AF4 and BCR-ABL positive ALL. These patients have a poor outcome with contemporary therapy and rapid identification facilitates timely allocation to innovative treatment programs.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Oncogénicas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , ARN Mensajero/biosíntesis , Niño , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Transcripción GenéticaRESUMEN
Here we show that exposure of human B-cell precursors to gamma-rays stimulates the enzymatic activity of the Src protooncogene family protein tyrosine kinase LYN. LYN activation in irradiated cells is not triggered by DNA damage or a nuclear signal since gamma-rays effectively stimulated LYN kinase in enucleated B-cell precursors as well. LYN activation in irradiated cells was abrogated by presence of the OH* radical scavenger dimethylsulfoxide and exposure of intact or enucleated B-cell precursors to chemically generated OH* radicals instead of gamma-rays also triggered LYN kinase activation and enhanced tyrosine phosphorylation of multiple electrophoretically distinct protein substrates. Thus, OH* radicals appear to be both mandatory and sufficient for radiation-induced LYN kinase activation in irradiated B-cell precursors. We further present evidence which indicates that OH* radicals activate LYN by a novel mechanism which involves disruption of inactive LYN-LYN homodimers and monomerization of the LYN kinase after proteolytic degradation of a putative LYN-associated adapter protein through a cytoplasmic TPCK-sensitive chymotrypsin-like protease following its oxidation. LYN kinase plays a pivotal role in initiation of signal cascades that affect the proliferation, differentiation, and survival of B-cell precursors. Our results prompt the hypothesis that a growth regulatory balance might be altered in human B-cell precursors by radiation-induced stimulation of LYN kinase.
Asunto(s)
Linfocitos B/efectos de la radiación , Radical Hidroxilo/metabolismo , Familia-src Quinasas/metabolismo , Linfocitos B/enzimología , Linfocitos B/inmunología , Línea Celular , Activación Enzimática , Rayos gamma , Humanos , Fosfotirosina/análisis , Inhibidores de Proteasas/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Clorometilcetona de Tosilfenilalanila/farmacología , Células Tumorales Cultivadas , Familia-src Quinasas/efectos de la radiaciónRESUMEN
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.
Asunto(s)
Linfocitos B/enzimología , Proteína Quinasa CDC2/metabolismo , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos B/citología , Linfocitos B/efectos de la radiación , Sitios de Unión/genética , Proteína Quinasa CDC2/química , Proteína Quinasa CDC2/genética , Reparación del ADN , Fase G2/efectos de la radiación , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tirosina/química , Tirosina/efectos de la radiación , Familia-src Quinasas/química , Familia-src Quinasas/genéticaRESUMEN
A novel technique for measurement of plasma exudation in the skin is described. Transferrin labelled in vivo with indium-113m is used as a plasma tracer. The conversion electrons from 113mIn are detected with a polystyrene crystal mounted on a photomultiplier tube. Owing to the short range of the electrons in tissue, background radiation from tracer circulating in underlying tissue will be very small, allowing plasma exudation in the skin to be detected with a high signal to noise ratio. The characteristics of the detector system are described in model experiments using sheets of mylar to simulate soft tissue. The acute inflammatory response to histamine provocation was studied in guinea pig skin. A dose-related increase in count rate representing vasodilatation and plasma exudation was detected over the skin after histamine provocation. The electron radiation system appears suitable for detection of low levels of superficial radioactivity and for pathophysiological studies of the skin.
Asunto(s)
Electrones , Exudados y Transudados/metabolismo , Plasma/metabolismo , Piel/irrigación sanguínea , Animales , Cobayas , Radioisótopos de Indio , MasculinoRESUMEN
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.
Asunto(s)
Linfocitos B/efectos de la radiación , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular , Fase G2/efectos de la radiación , Células Madre Hematopoyéticas/efectos de la radiación , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/efectos de la radiación , Proteínas Nucleares , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Procesamiento Proteico-Postraduccional/efectos de la radiación , Linfocitos B/metabolismo , Linfocitos B/patología , Benzoquinonas , Proteína Quinasa CDC2/antagonistas & inhibidores , Activación Enzimática/efectos de la radiación , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Lactamas Macrocíclicas , Sustancias Macromoleculares , Factor Promotor de Maduración/fisiología , Proteínas de Neoplasias/antagonistas & inhibidores , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de la radiación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimología , Proteínas Tirosina Quinasas/fisiología , Quinonas/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Rifabutina/análogos & derivados , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
We have examined the plasma exudation response of inflammation in guinea pig skin by a noninvasive method and have evaluated the influence of vasodilatation. Indium radionuclides have been used to label plasma and blood and conversion electrons have been detected by an external detector. Transferrin (79,600 Da) was labeled by 111In or 113mIn in vivo and red blood cells were labeled by 111In in vitro. These tracers were given to separate groups of anesthetized guinea pigs and baseline activities were recorded from shaved skin surface areas. Skin prick tests with histamine and saline were performed and time-activity curves were recorded. The measurements with 111In-transferrin and 111In-labeled red blood cells demonstrated that histamine produced dose-dependent accumulation of plasma (up to a 6.5-fold increase) and blood (up to a 2.0-fold increase) in the skin. Hence, about one-third of accumulation of plasma induced by histamine may be explained by vasodilatation. With 113mIn-transferrin as plasma tracer greater effects of histamine were recorded, probably reflecting that the measurements also included deeper sections of the skin. We conclude that the intensity of accumulation of plasma in skin inflammation can be monitored by external detection of conversion electrons from 111In- and 113mIn-transferrin, and that the influence of vasodilatation can be estimated by detection of 111In-labeled red blood cells.
Asunto(s)
Dermatitis/fisiopatología , Exudados y Transudados , Piel/fisiopatología , Vasodilatación , Animales , Dermatitis/diagnóstico por imagen , Electrones , Eritrocitos/diagnóstico por imagen , Cobayas , Radioisótopos de Indio , Masculino , Cintigrafía , Piel/diagnóstico por imagenRESUMEN
The study of central nervous system (CNS) leukemia has been hampered by the lack of a suitable animal model. We report that severe combined immunodeficiency (SCID) mice invariably develop rapidly progressive fatal CNS leukemia within 3 weeks after intravenous injection of NALM-6 pre-B acute lymphoblastic leukemia (ALL) cells. Colonization of the dura mater and subarachnoid space, usually of the distal spinal cord with occasional extension into the Virchow-Robin spaces of blood vessels subjacent to the meninges, followed involvement of bone marrow in the skull, vertebrae, and, occasionally, the appendicular skeleton. Occult CNS leukemia was detectable by polymerase chain reaction amplification of human DNA as early as 8 days postinoculation of leukemia cells. We used this in vivo model of human CNS leukemia to examine the therapeutic efficacy and toxicity of intrathecally administered B43 (anti-CD19)-pokeweed antiviral protein (PAP), an anti-B-lineage ALL immunotoxin directed against the pan-B-cell antigen CD19/Bp95. Intrathecal therapy with B43 (anti-CD19)-PAP immunotoxin at nontoxic dose levels significantly improved survival of SCID mice and was superior to intrathecal methotrexate therapy.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Sistema Nervioso Central/patología , Inmunotoxinas/uso terapéutico , Infiltración Leucémica/tratamiento farmacológico , Meninges/patología , N-Glicosil Hidrolasas , Proteínas de Plantas/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD19 , Barrera Hematoencefálica , Médula Ósea/patología , Movimiento Celular , ADN de Neoplasias/análisis , Evaluación Preclínica de Medicamentos , Inmunotoxinas/administración & dosificación , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Inyecciones Espinales , Metotrexato/uso terapéutico , Ratones , Ratones SCID , Trasplante de Neoplasias , Células Neoplásicas Circulantes , Proteínas de Plantas/administración & dosificación , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Organismos Libres de Patógenos Específicos , Trasplante HeterólogoRESUMEN
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.