RESUMEN
Targeting the PI3K isoform p110δ against B cell malignancies is at the mainstay of PI3K inhibitor (PI3Ki) development. Therefore, we generated isogenic cell lines, which express wild type or mutant p110δ, for assessing the potency, isoform-selectivity and molecular interactions of various PI3Ki chemotypes. The affinity pocket mutation I777M maintains p110δ activity in the presence of idelalisib, as indicated by intracellular AKT phosphorylation, and rescues cell functions such as p110δ-dependent cell viability. Resistance owing to this substitution consistently affects the potency of p110δ-selective in contrast to most multi-targeted PI3Ki, thus distinguishing usually propeller-shaped and typically flat molecules. Accordingly, molecular dynamics simulations indicate that the I777M substitution disturbs conformational flexibility in the specificity or affinity pockets of p110δ that is necessary for binding idelalisib or ZSTK474, but not copanlisib. In summary, cell-based and molecular exploration provide comparative characterization of currently developed PI3Ki and structural insights for future PI3Ki design.
Asunto(s)
Neoplasias , Fosfatidilinositol 3-Quinasas , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Línea CelularRESUMEN
Cytokines are soluble protein factors with importance in intercellular communication and, as such, play pivotal roles in the pathogenesis of B cell malignancies. Evidence from in vitro cultures permitted us to choose example cytokines that bind to different biochemical receptor types. Activated malignant B cells or stromal fibroblasts and macrophages prominently secrete the chemokines CCL3 or CXCL12 and CXCL13, respectively. Apart from helper T cells, various cell types of the B cell lymphoma microenvironment are capable of producing the cytokines IL-4, IL-6, IL-10 and TNFα. Owing to its impact on the development of myeloid cells, CSF-1 is among important soluble factors in the B cell lymphoma microenvironment. Inhibitors of B cell receptor-associated kinases often act via the blockade of cytokine production, but also prevent cytokine effects, e.g., chemotaxis. Increments in blood levels in chronic lymphocytic leukemia patients compared to healthy donors and normalization upon treatment with ibrutinib can be explained by producing cell types and modulation of cytokine production observed in vitro.
RESUMEN
On the occasion of its recent approval for relapsed follicular lymphoma, we review the design and development of the pan-class I PI3K inhibitor copanlisib as a drug for the treatment of B-cell malignancies in comparison with other kinase inhibitors targeting B-cell-receptor signaling, in particular with strictly isoform-δ-selective idelalisib. In agreement with previously defined PI3K-inhibitor chemotypes, the 2,3-dihydroimidazo[1,2-c]quinazoline scaffold of copanlisib adopts a flat conformation in the adenine-binding pocket of the catalytic p110 subunit and further extends into a deeper-affinity pocket in contrast to idelalisib, the quinazoline moiety of which is accommodated in a newly created selectivity pocket. Copanlisib shows higher potency than other clinically developed PI3K inhibitors against all four class I isoforms, with approximately tenfold preference for p110α and p110δ. Owing to its potency and isoform profile, copanlisib exhibits cell-type-specific cytotoxicity against primary chronic lymphocytic leukemia cells and diffuse large B-cell lymphoma (DLBCL) cell lines at nanomolar concentrations. Moreover, copanlisib differs from idelalisib in regard to intravenous versus oral administration and weekly versus twice-daily dosing. In regard to adverse effects, intermittent intravenous treatment with copanlisib leads to fewer gastrointestinal toxicities compared with continuous oral dosing of idelalisib. In relapsed follicular lymphoma, copanlisib appears more effective and especially better tolerated than other targeted therapies. Copanlisib extends existing treatment options for this subtype of indolent non-Hodgkin lymphoma and also shows promising response rates in DLBCL, especially of the activated B-cell type.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Quinazolinonas/farmacología , Animales , Antineoplásicos/química , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/química , Purinas/química , Pirimidinas/química , Quinazolinas/química , Quinazolinonas/químicaRESUMEN
The antibody-dependent cell-mediated cytotoxicity (ADCC) of the anti-CD20 monoclonal antibodies (mAbs) rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI) was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 µM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antígenos CD20/metabolismo , Linfocitos B/efectos de los fármacos , Citotoxinas/farmacología , Células Asesinas Naturales/efectos de los fármacos , Receptores de Antígenos de Linfocitos B/metabolismo , Adenina/análogos & derivados , Anticuerpos Monoclonales Humanizados/farmacología , Benzamidas/farmacología , Línea Celular Tumoral , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Piperidinas , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Pirazinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Quinazolinonas/farmacología , Rituximab/farmacologíaRESUMEN
To elucidate their mechanism of action, inhibitors of Bruton tyrosine kinase (BTK) and resistant BTK mutants were employed to dissect target-dependent cellular functions. BTK-C481S and -T474I, expressed in Ramos and NALM-6 cells, maintained BTK auto-phosphorylation under treatment with ibrutinib or dasatinib, respectively, which showed only modest cytotoxicity. Retained activity of BTK-T474 partially rescued cell migration from inhibition by dasatinib. Importantly, resistant BTK mutants reconstituted B cell receptor-triggered chemokine secretion in the presence of corresponding inhibitors, demonstrating that BTK activity is connected with cell-intrinsic functions of malignant B cells with importance for their dialogue with the micro-environment.
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Linfocitos B/enzimología , Leucemia de Células B/genética , Linfoma de Células B/genética , Proteínas Tirosina Quinasas/genética , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa , Antineoplásicos , Quimiocinas/metabolismo , Quimiotaxis/efectos de los fármacos , Dasatinib/administración & dosificación , Dasatinib/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Humanos , Leucemia de Células B/enzimología , Leucemia de Células B/patología , Linfoma de Células B/enzimología , Linfoma de Células B/patología , Mutación , Fosforilación/efectos de los fármacos , Piperidinas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Pirazoles/administración & dosificación , Pirazoles/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Células Tumorales CultivadasAsunto(s)
Anticuerpos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Antígenos de Neoplasias/inmunología , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Tetraspaninas/inmunología , Células Tumorales CultivadasRESUMEN
Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/farmacología , Isoquinolinas/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Purinas/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Quinazolinonas/farmacología , Antineoplásicos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimiocina CXCL12/fisiología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Rituximab , Transducción de SeñalRESUMEN
UNLABELLED: Survival of chronic lymphocytic leukemia (CLL) cells depends on stimuli provided by a suitable microenvironment. The factors and mechanisms providing this growth support for CLL cells are not fully understood. We found that plasma levels of macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, were elevated in CLL patients. Therefore, we characterized the functional role of MIF in a CLL mouse model. For this purpose, we crossed Eµ-TCL1 mice with MIF knockout (MIF-/-) mice. The resulting TCL1+/wtMIF/ mice showed a delayed onset of leukemia, reduced splenomegaly and hepatomegaly, and a longer survival than TCL1+/wtMIFwt/wt controls. Immunohistochemical examination of the lymphoid organs showed that the numbers of macrophages were significantly reduced in the spleen and bone marrow of TCL1+/wtMIF/ mice compared with TCL1+/wtMIFwt/wt controls. Mechanistic studies in vitro revealed that the absence of MIF rendered CLL cells more susceptible to apoptosis. Accordingly, incubation with an anti-MIF antibody reduced the survival of CLL cells on a macrophage feeder layer. In addition, the migratory activity of TCL1+/wtMIF/ macrophages was decreased compared with TCL1+/wtMIFwt/wt macrophages. Taken together, our results provide evidence that MIF supports the development of CLL by enhancing the interaction of CLL cells with macrophages. KEY POINTS: Targeted deletion of the gene for macrophage migration inhibitory factor (MIF) delays development of chronic lymphocytic leukemia and prolongs survival in mice. MIF recruits leukemia-associated macrophages to spleen or liver.
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Linfocitos T CD8-positivos/inmunología , Comunicación Celular/inmunología , Oxidorreductasas Intramoleculares/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Factores Inhibidores de la Migración de Macrófagos/inmunología , Macrófagos/inmunología , Animales , Linfocitos T CD8-positivos/patología , Supervivencia Celular , Células Nutrientes , Humanos , Oxidorreductasas Intramoleculares/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Factores Inhibidores de la Migración de Macrófagos/genética , Macrófagos/patología , Ratones , Ratones Noqueados , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Células Tumorales CultivadasRESUMEN
A series of novel pyrrolo-allocolchicine derivatives (containing a 1-methyl-1H-indol-5-yl moiety replacing ringâ C) was synthesized. The tetracyclic ring system was constructed by Suzuki-Miyaura cross-coupling of a 1-methylindole-5-boronate with an ortho-iodo-dihydrocinnamic acid derivative and subsequent intramolecular Friedel-Crafts acylation. After reduction of the resulting ketone, the nitrogen functionality was introduced in a Mitsunobu-type reaction by using zinc azide followed by LiAlH(4) reduction. Structural assignments were supported by X-ray crystallography. The compounds synthesized were then tested against BJAB tumor cells and found to exhibit pronounced cytotoxic activity (proliferation inhibition and apoptosis induction). The ketone 24 b was even active at sub-nanomolar concentration. In addition, the antitumor potential of the compounds was confirmed by using B lymphoid cell lines.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral/química , Línea Celular Tumoral/efectos de los fármacos , Colchicina/análogos & derivados , Colchicina/síntesis química , Colchicina/farmacología , Indoles/química , Indoles/síntesis química , Colchicina/química , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Relación Estructura-ActividadRESUMEN
BACKGROUND: With sorafenib displaying the highest affinities for Flt3, VEGFR (vascular endothelial growth factor receptor) and Raf and dasatinib for Abl and Src kinases, the profiles of kinases targeted by these inhibitors differ strongly. MATERIALS AND METHODS: Dose-dependent effects of the inhibitors on freshly isolated chronic lymphocytic leukemia (CLL) cells were assessed as increased phosphatidylserine exposure. Inhibition by sorafenib and dasatinib of survival and anti-apoptotic signaling in CLL cells was examined by Western blot analysis. RESULTS: Sorafenib uniformly induced apoptosis in CLL lymphocytes with a concentration inhibiting by 50% (IC50) of 8 mM, whereas the response to dasatinib was heterogeneous with the onset of inhibition at submicromolar concentrations but with IC50 values below 25 mM in only a few samples. At the respective pharmacologically achievable plasma concentrations, the inhibitors showed more efficient apoptosis induction by sorafenib than by dasatinib and less than additive mutual enhancement in combination. Co-culture with the bone marrow stroma cell line HS-5 increased the viability of untreated CLL cells but did not protect from sorafenib-induced apoptosis. CONCLUSIONS: Sorafenib or dasatinib displayed sigmoidal or saturation-type dose-response relationships for apoptosis induction, which were uniform or highly divergent, respectively, among individual CLL samples and therefore might complement each other in their clinical potential for CLL.
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Bencenosulfonatos/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/fisiopatología , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridinas/administración & dosificación , Pirimidinas/administración & dosificación , Tiazoles/administración & dosificación , Dasatinib , Relación Dosis-Respuesta a Droga , Humanos , Dosificación Letal Mediana , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Sorafenib , Resultado del Tratamiento , Células Tumorales CultivadasRESUMEN
The effects of two CD20 antibodies, namely rituximab, the current standard for treatment of chronic lymphocytic leukaemia (CLL) in combination with chemotherapy, and GA101, a glyco-engineered type II antibody were compared on CLL cells ex vivo. Antibody-induced phosphatidylserine exposure was examined in isolated CLL cells. For a more comprehensive assessment of antibody-mediated cell killing including Fc-mediated mechanisms, B cell depletion from whole blood samples was monitored. Treatment with rituximab or GA101 reduced the average viability of isolated CLL cells by 6% or 11%, and the ratio of B to T cells in whole blood samples by 12% or 33%, respectively. Combination with GA101 enhanced the cytotoxicity of the chemotherapeutic agent chlorambucil on isolated CLL cells. CD20 surface expression on CLL cells correlated with GA101-induced B cell depletion, but not with direct cell death induction. Treatment of whole blood samples from CLL patients with a CpG-containing oligonucleotide increased CD20 expression on CLL cells and GA101-dependent B cell depletion. Despite the variable responses of individual CLL samples, the CLL cell depletion from whole blood by GA101 was consistently much stronger than by rituximab, which argues for clinical investigation of GA101 in CLL patients.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antígenos CD20/inmunología , Antígenos CD20/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Linfocitos B/inmunología , Linfocitos B/patología , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Depleción Linfocítica/métodos , Masculino , Persona de Mediana Edad , Rituximab , Células Tumorales CultivadasAsunto(s)
Antineoplásicos/uso terapéutico , Autofagia/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Pirimidinas/uso terapéutico , Tiazoles/uso terapéutico , Antineoplásicos/farmacología , Dasatinib , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Pirimidinas/farmacología , Tiazoles/farmacologíaRESUMEN
MicroRNAs (miRNA) play a key role in cellular regulation and, if deregulated, in the development of neoplastic disorders including chronic lymphocytic leukemia (CLL). RNAs from primary cells of 50 treatment-naive CLL patients and peripheral B cells of 14 healthy donors were applied to miRNA expression profiling using bead chip technology. In CLL cells, a set of 7 up- and 19 down-regulated miRNAs was identified. Among the miRNAs down-regulated in CLL cells, 6 of 10 miRNA promoters examined showed gain of methylation compared with normal B-cell controls. Subsequent target prediction of deregulated miRNAs revealed a highly significant binding prediction at the 3' untranslated region of the pleomorphic adenoma gene 1 (PLAG1) oncogene. Luciferase reporter assays including site-directed mutagenesis of binding sites revealed a significant regulation of PLAG1 by miR-181a, miR-181b, miR-107, and miR-424. Although expression of PLAG1 mRNA was not affected, PLAG1 protein expression was shown to be significantly elevated in CLL cells compared with the levels in healthy donor B cells. In summary, we could demonstrate disruption of miRNA-mediated translational control, partly due to epigenetic transcriptional silencing of miRNAs, with subsequent overexpression of the oncogenic transcription factor PLAG1 as a putative novel mechanism of CLL pathogenesis.
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Proteínas de Unión al ADN/biosíntesis , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Leucemia Linfocítica Crónica de Células B/metabolismo , MicroARNs/metabolismo , Proteínas Oncogénicas/biosíntesis , Estabilidad del ARN , ARN Neoplásico/metabolismo , Regiones no Traducidas 3'/genética , Linfocitos B/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Masculino , MicroARNs/genética , Proteínas Oncogénicas/genética , Regiones Promotoras Genéticas/genética , ARN Neoplásico/genética , Transcripción Genética/genéticaRESUMEN
Src family kinases (SFKs) were described to be overexpressed in chronic lymphocytic leukemia (CLL). We wished to examine the effects of the Src and Abl kinase inhibitor dasatinib on the intracellular signaling and survival of CLL cells. Dasa-tinib showed a dose- and time-dependent reduction of global tyrosine phosphorylation and of activating phosphotyrosine levels of SFKs. Treatment with 100 nM dasatinib led to decreased levels of the activated, phosphorylated forms of Akt, Erk1/2, and p38, and induced PARP cleavage through caspase activity. In Mec1 and JVM-3 cell lines, dasatinib increased p53 protein levels and inhibited proliferation. In freshly isolated CLL cells, dasatinib reduced the expression of Mcl-1 and Bcl-x(L). Combination of 5 microM dasatinib and fludarabine increased the apoptosis induction of each by approximately 50%. In 15 primary CLL samples, cells with unmutated immunoglobulin variable heavy chain (IgV(H)) genes were more sensitive to dasatinib than those with mutated IgV(H) genes (P = .002). In summary, dasatinib shows potent inhibitory effects on the survival of CLL cells in vitro, most prominently in samples obtained from patients with unfavorable prognostic features.
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Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Pirimidinas/farmacología , Tiazoles/farmacología , Caspasas/metabolismo , Dasatinib , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Cinética , Leucemia Linfocítica Crónica de Células B/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Células Tumorales Cultivadas , Vidarabina/análogos & derivados , Vidarabina/farmacología , Proteína bcl-X/efectos de los fármacosRESUMEN
The constitutive tyrosine kinase activity of the BCR-ABL fusion protein plays a crucial role in the pathogenesis of chronic myeloid leukemia and promotes growth factor-independent survival of hematopoietic cells. In 32D cells, expression levels of retrovirally transduced BCR-ABL were positively correlated with the levels of the cell cycle regulator protein p21, and this upregulation of p21 expression depended on the kinase activity of BCR-ABL. To assess the role of p21 on BCR-ABL-positive hematopoietic cells, we compared proliferation and drug-induced apoptosis in bone marrow (BM) cells from wild-type and p21 knockout mice after retroviral transfer of the BCR-ABL fusion gene. As compared with wild-type cells, p21 knockout cells showed increased proliferation, suggesting that p21 acted as an attenuator of BCR-ABL-mediated cell proliferation. In marked contrast, deletion of p21 promoted apoptosis induction by imatinib and taxol in BCR-ABL-transformed BM cells. These findings demonstrate that p21 has a dual function in BCR-ABL-transformed murine BM cells: It attenuates the effects of two apparently opposed phenomena such as BCR-ABL-mediated cell proliferation and drug-induced apoptosis. This dual function of p21 calls for a cautious evaluation of the suitability of p21 as a secondary target in anticancer therapy.
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Apoptosis/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Paclitaxel/farmacología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Moduladores de Tubulina/farmacología , Animales , Benzamidas , Línea Celular Transformada , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Fusión bcr-abl , Células Madre Hematopoyéticas , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Ratones , Ratones Noqueados , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Retroviridae , Transducción GenéticaRESUMEN
The receptor tyrosine kinase (RTK) Flt3 is expressed in early hematopoietic progenitor cells and stimulates their growth. Due to frequent mutations in the Flt3 gene in patients with acute myeloid leukemia (AML), Flt3 is regarded as a potential therapeutic target, but the underlying mechanisms are still poorly understood. Therefore, we investigated interactions of Flt3 and some Src family tyrosine kinases (SFKs), which are expressed predominantly or exclusively in hematopoietic cells and known to be involved in signal transduction by various RTKs. Employing sets of wt and mutant Flt3 and Hck, we analyzed protein binding as well as Flt3 phosphorylation and maturation in HEK-293 cells cotransfected with expression constructs encoding both binding partners. Kinase-inactive Hck-K269R was recruited to phosphotyrosine residues located in the juxtamembrane (JM) region of activated Flt3 via its SH2 domain. Several of the JM domain tyrosines were phophorylated by Hck and other SFKs. As apparent from the distribution of mature and hypoglycosylated Flt3, SFKs interfered with Flt3 maturation in a kinase-dependent manner. Together, these findings show a complex role of SFKs in Flt3 signaling and reveal a new function of SFKs in the maturation of RTKs.
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Procesos de Crecimiento Celular/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/metabolismo , Familia-src Quinasas/metabolismo , Línea Celular , Hematopoyesis/fisiología , Humanos , Proteínas Proto-Oncogénicas c-hck/metabolismo , TransfecciónRESUMEN
The aim of the Evaluation-guided Development of new In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R(2) = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that the development within the EDIT target organ programme should initially be focused on the nervous system.
Asunto(s)
Toxicología/métodos , Biotransformación , Barrera Hematoencefálica , Daño del ADN , Humanos , Técnicas In Vitro , Absorción Intestinal , Riñón/metabolismo , Unión Proteica , Relación Estructura-Actividad CuantitativaRESUMEN
The metabolically competent human lymphoblastoid cell line MCL-5 was treated with a panel of mutagens to assess the induction of DNA damage. Treatment effects were observed by monitoring cell proliferation and by single-cell gel electrophoresis (SCGE). The direct-acting mutagens benzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), as well as pro-mutagens requiring metabolic activation, i.e. benzo[a]pyrene (BaP), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 4-N-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and cigarette-smoke condensate (CSC), were assayed by SCGE. Assay schemes were adapted for the MCL-5 cell line and for low levels of strand break induction, by inclusion of the DNA synthesis inhibitors cytosine arabinoside and hydyroxyurea, and by extending the electrophoresis time. For all mutagens tested, dose-dependent increases of median and average tail moment values among 50 nucleoids per slide were observed. The determining factors for selecting the treatment doses for mutation-induction experiments were the solubility of BaP and PhIP in the exposure medium, and the cytotoxicity exhibited by BPDE, MNNG and CSC. Induction of DNA strand breaks was obtained at mutagen concentrations permitting sufficient cell proliferation, except in the case of MNNG.