RESUMEN
Although tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of chronic myeloid leukemia (CML), the ability of TKIs to eradicate CML remains uncertain and patients must continue TKI therapy for indefinite periods. In this study, we performed whole-exome sequencing to identify somatic mutations in 24 patients with newly diagnosed chronic phase CML who were registered in the JALSG CML212 study. We identified 191 somatic mutations other than the BCR-ABL1 fusion gene (median 8, range 1-17). Age, hemoglobin concentration and white blood cell counts were correlated with the number of mutations. Patients with mutations ⩾6 showed higher rate of achieving major molecular response than those<6 (P=0.0381). Mutations in epigenetic regulator, ASXL1, TET2, TET3, KDM1A and MSH6 were found in 25% of patients. TET2 or TET3, AKT1 and RUNX1 were mutated in one patient each. ASXL1 was mutated within exon 12 in three cases. Mutated genes were significantly enriched with cell signaling and cell division pathways. Furthermore, DNA copy number analysis showed that 2 of 24 patients had uniparental disomy of chromosome 1p or 3q, which disappeared major molecular response was achieved. These mutations may play significant roles in CML pathogenesis in addition to the strong driver mutation BCR-ABL1.
Asunto(s)
Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Histona Demetilasas/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Factores de Edad , Variaciones en el Número de Copia de ADN/genética , Resistencia a Antineoplásicos/genética , Epigénesis Genética/genética , Femenino , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Recuento de Leucocitos , Masculino , Mutación , Inhibidores de Proteínas Quinasas/administración & dosificación , Transducción de Señal , Secuenciación del ExomaRESUMEN
Somatic mutation of RUNX1 is implicated in various hematological malignancies, including myelodysplastic syndrome and acute myeloid leukemia (AML), and previous studies using mouse models disclosed its critical roles in hematopoiesis. However, the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder (FPD)/AML is an autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. By in vitro differentiation assays, FPD-iPSCs were clearly defective in the emergence of hematopoietic progenitors and differentiation of megakaryocytes, and overexpression of wild-type (WT)-RUNX1 reversed most of these phenotypes. We further demonstrated that overexpression of mutant-RUNX1 in WT-iPSCs did not recapitulate the phenotype of FPD-iPSCs, showing that the mutations were of loss-of-function type. Taken together, this study demonstrated that haploinsufficient RUNX1 allele imposed cell-intrinsic defects on hematopoietic differentiation in human experimental settings and revealed differential impacts of RUNX1 dosage on human and murine megakaryopoiesis. FPD-iPSCs will be a useful tool to investigate mutant RUNX1-mediated molecular processes in hematopoiesis and leukemogenesis.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Hematopoyesis/genética , Células Madre Pluripotentes Inducidas/metabolismo , Leucemia Mieloide Aguda/genética , Mutación , Animales , Trastornos de las Plaquetas Sanguíneas/patología , Diferenciación Celular/genética , Análisis Mutacional de ADN , Femenino , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Inmunofenotipificación , Células Madre Pluripotentes Inducidas/patología , Leucemia Mieloide Aguda/patología , Masculino , Megacariocitos/metabolismo , Megacariocitos/patología , Ratones , Linaje , FenotipoRESUMEN
OBJECTIVE: Biomarkers of disease activity in lupus nephritis (LN) are needed. Ideally, such biomarkers would be capable of detecting early sub-clinical disease and could be used to gauge response to therapy, thus obviating the need for serial renal biopsies. Much of the focus in the search for LN biomarkers has been on the measurement of urinary chemokines and cytokines in LN patients. However, these have yet to be widely implemented in clinical practice. Kidney injury molecule-1 (Kim-1) is expressed in damaged tubules, but whether urinary (u) and tubular (t)-Kim-1 could serve as a biomarker of active LN is unknown. To investigate the disease activity and histological findings in LN, we evaluated u-Kim-1 levels and t-Kim-1 cells in patients with systemic lupus erythematosus (SLE). METHOD: We measured u-Kim-1 levels and stained t-Kim-1 expression in 57 patients with LN using an ELISA and immunohistochemistry staining. Patients were classified into two groups (active LN, n = 37; inactive LN, n = 20) based on the presence of active renal disease according to the renal SLE disease activity index. correlations of clinical, laboratory data, and histological findings with urinary and t-Kim-1 expression were assessed. RESULT: The u-Kim-1 levels were significantly correlated with the expression of t-Kim-1 (R = 0.64; P = 0.004) in the SLE patients. The active LN patients exhibited elevated u-Kim-1 levels compared to the inactive LN patients. The number of t-Kim-1 cells was also correlated with histological findings (both glomerular and interstitial inflammation). The u-Kim-1 levels were also correlated with proteinuria and tubular damage in the active LN group. The number of t-Kim-1 cells at baseline was significantly correlated with the estimated glomerular filtration rate (R = 0.72; P = 0.005) and serum creatinine (R = 0.53; P = 0.005) after 6-8 months of treatment. CONCLUSION: These data suggest the potential use of the u-Kim-1 levels to screen for active LN and for the estimation of t-Kim-1 expression in renal biopsies to predict renal damage, ongoing glomerular nephritis and tubulointerstitial inflammation, and tubular atrophy.
Asunto(s)
Nefritis Lúpica/orina , Glicoproteínas de Membrana/orina , Adulto , Biomarcadores/orina , Femenino , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Masculino , Receptores ViralesRESUMEN
Loss-of-function mutations of RUNX1 have been found in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs). Although several reports have suggested roles for RUNX1 as a tumor suppressor, its precise function remains unknown. Because gene alterations of RUNX1 by themselves do not lead to the development of leukemia in mouse models, additional mutation(s) would be required for leukemia development. Here, we report that the C-terminal deletion mutant of RUNX1, RUNX1dC, attenuates DNA-damage repair responses in hematopoietic stem/progenitor cells. γH2AX foci, which indicate the presence of DNA double-strand breaks, were more abundantly accumulated in RUNX1dC-transduced lineage(-)Sca1(+)c-kit(+) (LSK) cells than in mock-transduced LSK cells both in a steady state and after γ-ray treatment. Expression profiling by real-time -PCR array revealed RUNX1dC represses the expression of Gadd45a, a sensor of DNA stress. Furthermore, bone marrow cells from MDS/AML patients harboring the RUNX1-C-terminal mutation showed significantly lower levels of GADD45A expression compared with those from MDS/AML patients with wild-type RUNX1. As for this mechanism, we found that RUNX1 directly regulates the transcription of GADD45A and that RUNX1 and p53 synergistically activate the GADD45A transcription. Together, these results suggest Gadd45a dysfunction due to RUNX1 mutations can cause additional mutation(s) required for multi-step leukemogenesis.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Daño del ADN , Reparación del ADN , Células Madre Hematopoyéticas/metabolismo , Mutación , Animales , Proteínas de Ciclo Celular/genética , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Síndromes Mielodisplásicos/genética , Proteínas Nucleares/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción GenéticaRESUMEN
Although there is growing evidence that estrogens promote tumor progression in epithelial ovarian cancer, the molecular mechanisms accounting for this are still unclear. Selective estrogen receptor modulators (SERMs) mimic estrogen action in certain tissues while opposing it in others. The molecular mechanisms of the effects of SERMs such as raloxifene on the tumor progression of epithelial ovarian cancer are also still unclear. Here, we show that various genomic actions of estrogen differ from those of raloxifene in human ovarian cancer cell lines expressing estrogen receptor alpha (ERalpha). 17beta-Estradiol (E2) induced the gene expression of c-Myc and IGF-1 and increased the binding of ERalpha to the AP1 site of the promoters of c-Myc and IGF-1. ERalpha silencing abolished the E2-stimulated c-Myc expression. E2 induced the recruitment of co-activators such as SRC-1, SRC-3 and CBP to the promoters of c-Myc and IGF-1, and SRC-1 silencing abolished both the E2-stimulated c-Myc expression and cell-cycle progression. In contrast, although raloxifene increased the binding of ERalpha to the AP1 site of the promoters of c-Myc and IGF-1, raloxifene had no effect on the gene expression of c-Myc or IGF-1. Raloxifene induced the recruitment of co-repressors such as HDAC2, N-CoR and SMRT to the promoter of IGF-1. Thus, the difference between the genomic actions exerted by estrogen and raloxifene in human ovarian cancer cell lines expressing ERalpha appear to be dependent on the recruitment of co-regulators.
Asunto(s)
Estrógenos/fisiología , Genoma Humano/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Clorhidrato de Raloxifeno/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Línea Celular Tumoral , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/genética , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Growth, survival and differentiation of hematopoietic cells are regulated by the interaction between hematopoietic growth factors and their receptors. While the defect in this interaction results in an insufficient hematopoiesis, the aberrantly elevated activation leads to the transformation of hematopoietic cells. The constitutive active mutations of receptor tyrosine kinase, such as c-Kit platelet-derived growth factor receptor (PDGFR) or fins-like tyrosine kinase 3 (Flt3), play a major role in the development of hematopoietic neoplasia. The constitutive activation is provoked by several mechanisms, such as making fusion genes by chromosomal translocations, or various mutations involving regulatory regions of the receptor. The chromosomal translocation brings the receptor intracytoplasmic domain juxtaposed to an unrelated molecule which has dimerization or multimerization motif, resulting in the constitutive dimerization of the receptor. The missense, insertion or deletion mutations in the regulatory regions, such as juxtamembrane domain, activation loop and extracellular domain, cause constitutive activation by releasing the respective auto-inhibitory functions of each regulatory region. Constitutive active receptors generate different signals quantitatively and qualitatively from wild type receptor, which mediate the oncogenic phenotype. Given the frequent involvement of constitutive active receptor tyrosine kinase in hematopoietic malignancies, targeted inhibitions of active tyrosine kinase and downstream aberrant signaling are rapidly developing novel therapeutic modality with much promise.
Asunto(s)
Leucemia/enzimología , Proteínas Tirosina Quinasas Receptoras/fisiología , Animales , Humanos , Leucemia/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Tirosina Quinasa 3 Similar a fmsRESUMEN
During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3-dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.
Asunto(s)
Ciclo Celular/fisiología , Eritropoyesis/fisiología , Regulación Enzimológica de la Expresión Génica , Células Madre Hematopoyéticas/fisiología , Megacariocitos/fisiología , Poliploidía , Proteínas Quinasas/genética , Animales , Aurora Quinasa A , Aurora Quinasas , Células de la Médula Ósea/citología , División Celular , Línea Celular , Células Cultivadas , Replicación del ADN , Femenino , Genes ras , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interleucina-3/farmacología , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ésteres del Forbol/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Proteínas Recombinantes/farmacología , Factor de Células Madre/farmacología , Trombopoyetina/farmacología , Transcripción GenéticaRESUMEN
The Escherichia coli beta-glucuronidase (GUS) was evolved in vitro to catalyze the hydrolysis of a beta-galactoside substrate 500 times more efficiently (k(cat)/K(m)) than the wild-type, with a 52 million-fold inversion in specificity. The amino acid substitutions that recurred among 32 clones isolated in three rounds of DNA shuffling and screening were mapped to the active site. The functional consequences of these mutations were investigated by introducing them individually or in combination into otherwise wild-type gusA genes. The kinetic behavior of the purified mutant proteins in reactions with a series of substrate analogues show that four mutations account for the changes in substrate specificity, and that they are synergistic. An evolutionary intermediate, unlike the wild-type and evolved forms, exhibits broadened specificity for substrates dissimilar to either glucuronides or galactosides. These results are consistent with the "patchwork" hypothesis, which postulates that modern enzymes diverged from ancestors with broad specificity.
Asunto(s)
Evolución Molecular Dirigida , Escherichia coli/enzimología , Glucuronidasa/química , Glucuronidasa/metabolismo , beta-Galactosidasa/química , beta-Galactosidasa/metabolismo , Escherichia coli/genética , Galactósidos/química , Galactósidos/metabolismo , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Glucuronidasa/genética , Glucuronidasa/aislamiento & purificación , Humanos , Cinética , Modelos Moleculares , Mutación/genética , Conformación Proteica , Ingeniería de Proteínas , Especificidad por Sustrato , beta-Galactosidasa/genética , beta-Galactosidasa/aislamiento & purificaciónRESUMEN
Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD-mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways. (Blood. 2000;96:3907-3914)
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Leucemia Mieloide/fisiopatología , Proteínas de la Leche , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/farmacología , Enfermedad Aguda , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , División Celular/efectos de los fármacos , Células Clonales/citología , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Femenino , Humanos , Leucemia Mieloide/genética , Ratones , Ratones Endogámicos C3H , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Células Mieloides/efectos de los fármacos , Células Mieloides/fisiología , Neoplasias Experimentales/mortalidad , Neoplasias Experimentales/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Factor de Transcripción STAT5 , Transducción de Señal/efectos de los fármacos , Secuencias Repetidas en Tándem/genética , Transactivadores/metabolismo , Transactivadores/fisiología , Transfección , Células Tumorales Cultivadas , Tirosina Quinasa 3 Similar a fms , Proteínas ras/metabolismo , Proteínas ras/fisiologíaRESUMEN
Lineage-specific transcription factors play crucial roles in the development of hematopoietic cells. In a previous study, it was demonstrated that Ras activation was involved in thrombopoietin-induced megakaryocytic differentiation. In this study, constitutive Ras activation by H-ras(G12V) evoked megakaryocytic maturation of erythroleukemia cell lines F-36P and K562, but not of myeloid cell line 32D cl3 that lacks GATA-1. However, the introduction of GATA-1 led to reprogramming of 32D cl3 toward erythrocytic/megakaryocytic lineage and enabled it to undergo megakaryocytic differentiation in response to H-ras(G12V). In contrast, the overexpression of PU.1 and c-Myb changed the phenotype of K562 from erythroid to myeloid/monocytic lineage and rendered K562 to differentiate into granulocytes and macrophages in response to H-ras(G12V), respectively. In GATA-1-transfected 32D cl3, the endogenous expression of PU.1 and c-Myb was easily detectable, but their activities were reduced severely. Endogenous GATA-1 activities were markedly suppressed in PU.1-transfected and c-myb-transfected K562. As for the mechanisms of these reciprocal inhibitions, GATA-1 and PU.1 were found to associate through their DNA-binding domains and to inhibit the respective DNA-binding activities of each other. In addition, c-Myb bound to GATA-1 and inhibited its DNA-binding activities. Mutant GATA-1 and PU.1 that retained their own transcriptional activities but could not inhibit the reciprocal partner were less effective in changing the lineage phenotype of 32D cl3 and K562. These results suggested that GATA-1 activities may be crucial for Ras-mediated megakaryocytic differentiation and that its activities may be regulated by the direct interaction with other lineage-specific transcription factors such as PU.1 and c-Myb.
Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/fisiología , Células Madre Hematopoyéticas/citología , Megacariocitos/citología , Factores de Transcripción/fisiología , Proteínas ras/fisiología , Animales , Células de la Médula Ósea , Línea Celular , Proteínas de Unión al ADN/genética , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Expresión Génica , Genes ras/genética , Granulocitos/citología , Humanos , Leucemia Eritroblástica Aguda , Macrófagos/citología , Ratones , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Recombinantes , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Factores de Transcripción/genética , Transfección , Células Tumorales Cultivadas , Proteínas ras/genéticaRESUMEN
A new human myeloma cell line, OPM-6, was established from the peripheral blood of a patient with advanced IgG-kappa plasma cell leukemia. Cytogenetic and phenotypic analysis confirmed that the cells were derived from the patient's leukemic cells. Insulin-like growth factor-1 (IGF-1) acts as an autocrine growth factor in these cells. In addition, OPM-6 cells were particularly sensitive to dexamethasone (DEX), when endogenous IGF-1 was blocked. Under these conditions, >95% of the DEX-treated cells died within 36 h. Therefore, OPM-6 represents a potentially powerful tool for the analysis of the molecular mechanisms of DEX-induced apoptosis, because it is possible to easily analyze the direct effects of DEX using this system. Using this culture system of OPM-6, we demonstrated that the treatment with DEX plus a monoclonal antibody to the human IGF-1 receptor (alphaIGF-1R) leads to the down-regulation of the gene expression of Bcl-xL, an antiapoptotic gene, and the activation of CPP32 during this apoptotic process. IFN-alpha as well as IL-6 prevented DEX plus alphaIGF-1R-induced apoptosis, and this prevention was blocked by the mitogen-activated protein kinase kinase inhibitor, PD098059, or the phosphatidylinositol 3-kinase inhibitor, wortmannin. Therefore, both IL-6 and IFN-alpha blocked DEX plus alphaIGF-1R-induced apoptosis through activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.
Asunto(s)
Apoptosis/efectos de los fármacos , Dexametasona/antagonistas & inhibidores , Interferón Tipo I/farmacología , Interleucina-6/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mieloma Múltiple/patología , Fosfatidilinositol 3-Quinasas/fisiología , Células Tumorales Cultivadas/patología , Anciano , Apoptosis/genética , Caspasa 3 , Caspasas/metabolismo , Dexametasona/toxicidad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Mieloma Múltiple/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor IGF Tipo 1/fisiología , Receptor de Interferón alfa y beta , Receptores de Interferón/fisiología , Receptores de Interleucina-6/fisiología , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Tyrosine kinase oncoproteins cause simultaneous activation of multiple intracellular signaling pathways. However, the precise mechanisms by which individual pathways induce oncogenesis are not well understood. We have investigated the roles of individual signaling pathways in v-Src-dependent cell growth and survival by inhibiting one particular pathway. v-Src induced constitutive activation of signal transducers and activators of transcription 3 (STAT3), phosphatidylinositol 3-kinase, and Ras in murine Ba/F3 cells and led to factor-independent proliferation. Dominant-negative mutants of STAT3 (STAT3D) and phosphatidylinositol 3-kinase (Deltap85) inhibited v-Src-dependent growth by approximately 60 and approximately 40%, respectively. Moreover, dominant-negative Ras (N17) induced severe apoptosis, which was accompanied by down-regulation of Bcl-2 and activation of caspase-3. Although cells overexpressing Bcl-2 or caspase-3 inhibitors remained viable even when N17 was expressed, the growth was reduced by approximately 85%. During N17- and STAT3D-induced growth suppression, expression of cyclin D2, cyclin D3, c-myc, and c-fos was suppressed by N17, whereas that of cyclin D2, cyclin E, and c-myc was suppressed by STAT3D. Thus, v-Src-activated Ras and STAT3 are involved in distinct but partly overlapping transcriptional regulation of cell cycle regulatory molecules. These results suggest that the full oncogenic activity of v-Src requires simultaneous activation of multiple signalings, in which Ras is particularly required for survival.
Asunto(s)
Transformación Celular Neoplásica , Proteínas de Unión al ADN/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Transactivadores/metabolismo , Proteínas ras/metabolismo , Animales , Apoptosis , Linfocitos B , División Celular , Supervivencia Celular , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Interleucina-3/farmacología , Ratones , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Factor de Transcripción STAT3 , Transducción de Señal , Células Madre , Transactivadores/genética , Proteínas ras/genéticaRESUMEN
Cell migration requires a dynamic interaction between the cell, its substrate, and the cytoskeleton-associated motile apparatus. Integrin-associated protein (IAP)/CD47 is a 50-kd cell surface protein that is physically associated with beta3 integrins and that modulates the functions of beta3 integrins in various cells. However, in B-lymphocytes that express beta1 integrins but few beta3 integrins, the roles of IAP/CD47 remain to be determined. Cross-linking of IAP/CD47 by the immobilized anti-IAP/CD47 monoclonal antibody (mAb) B6H12, but not 2D3, produced signals to promote polarization with lamellipodia, a characteristic morphology during leukocyte migration, in pre-B and mature B-cell lines (BALL, Nalm6, ONHL-1, Daudi), but not in myeloma cell lines (RPMI8226, OPM-2). In the presence of the immobilized fibronectin (FN), soluble B6H12 could increase the rate of the polarization and activate migratory activity of BALL cells to FN in a transwell filter assay. Furthermore, the dominant-negative form of CDC42 completely blocked B6H12-induced morphologic and functional changes without inhibiting phorbol 12-myristate 13-acetate-induced spreading on FN in BALL cells, whereas the dominant-negative form of Rac1 inhibited all these changes. These findings demonstrate that in B-lymphocytes, IAP/CD47 may transduce the signals to activate the migratory activity, in which CDC42 may be specifically involved, and that IAP/CD47 shows synergistic effect with alpha4beta1 on B-cell migration. These findings would provide new insight into the role of IAP/CD47 on B-cell function.
Asunto(s)
Antígenos CD/fisiología , Linfocitos B/fisiología , Proteínas Portadoras/fisiología , Proteína de Unión al GTP cdc42/metabolismo , Anticuerpos Monoclonales/farmacología , Linfocitos B/citología , Linfoma de Burkitt , Antígeno CD47 , Polaridad Celular , Reactivos de Enlaces Cruzados , Fibronectinas/fisiología , Humanos , Integrina beta1/fisiología , Integrina beta3 , Glicoproteínas de Membrana Plaquetaria/fisiología , Receptores de Fibronectina/fisiología , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección , Células Tumorales CultivadasRESUMEN
At the late phase of megakaryocytopoiesis, megakaryocytes undergo endomitosis, which is characterized by DNA replication without cell division. Although a number of cell cycle regulatory molecules have been identified, the precise roles of these molecules in megakaryocytic endomitosis are largely unknown. In a human interleukin-3-dependent cell line transfected with the thrombopoietin (TPO) receptor c-mpl (F-36P-mpl), either treatment with TPO or the overexpression of activated ras (Ha-Ras(G12V)) induced megakaryocytic maturation with polyploid formation. We found that TPO stimulation or Ha-Ras(G12V) expression led to up-regulation of cyclin D1, cyclin D2, and cyclin D3 expression. In addition, expression levels of cyclin A and cyclin B were reduced during the total course of both TPO- and Ha-Ras(G12V)-induced megakaryocytic differentiation, thereby leading to decreased cdc2 kinase activity. Neither the induced expression of cyclin D1, cyclin D2, or cyclin D3 nor the expression of a dominant negative form of cdc2 alone could induce megakaryocytic differentiation of F-36P-mpl cells. In contrast, overexpression of dominant negative cdc2 together with cyclin D1, cyclin D2, or cyclin D3 facilitated megakaryocytic differentiation in the absence of TPO. These results suggest that both D-type cyclin expression and decreased cdc2 kinase activity may participate in megakaryocytic differentiation.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclinas/fisiología , Células Madre Hematopoyéticas/citología , Megacariocitos/citología , Trombopoyetina/metabolismo , Ciclo Celular/efectos de los fármacos , Diferenciación Celular , Ciclina D , Ciclina D1/metabolismo , Ciclina D2 , Ciclina D3 , Ciclinas/metabolismo , ADN Complementario/metabolismo , Citometría de Flujo , Genes Dominantes , Humanos , Isopropil Tiogalactósido/farmacología , Plásmidos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Recombinantes/metabolismo , Trombopoyetina/farmacología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Cytokines exert pleiotropic effects on target cells in a manner dependent on the cell type or stage of differentiation. To determine how instinctive cell properties affect biological effects of cytokine, we introduced an erythroid/megakaryocyte lineage-specific transcription factor, GATA-1, into a murine myeloid cell line M1, which is known to undergo macrophage differentiation in response to interleukin 6 (IL-6). Overexpression of GATA-1 changed the phenotype of M1 cells from myeloid to megakaryocytic lineage. Furthermore, GATA-1 blocked both IL-6-induced macrophage differentiation and apoptosis of M1 cells. Although STAT3 is essential for IL-6-induced macrophage differentiation of M1 cells, GATA-1 had little or no effect on tyrosine phosphorylation, DNA binding, and transcriptional activities of STAT3 in Western blot analysis, electropholic mobility shift assay (EMSA), and luciferase assays. During IL-6-induced macrophage differentiation of M1 cells, IL-6 down-regulated cyclin D1 expression and induced p19(INK4D) expression, leading to reduction in cdk4 activities. In contrast, sustained expression of cyclin D1 and a significantly lesser amount of p19(INK4D) induction were observed in IL-6-treated M1 cells overexpressing GATA-1. Furthermore, although bcl-2 expression was severely reduced by IL-6 in M1 cells, it was sustained in GATA-1-introduced M1 cells during the culture with IL-6. Both IL-6-induced macrophage differentiation and apoptosis were significantly abrogated by coexpression of cyclin D1 and bcl-2, whereas overexpressions of cyclin D1 or bcl-2 inhibited only differentiation or apoptosis, respectively. These results suggested that GATA-1 may not only reprogram the lineage phenotype of M1 cells but also disrupt the biologic effects of IL-6 through the sustained expression of cyclin D1 and bcl-2. (Blood. 2000;95:1264-1273)
Asunto(s)
Apoptosis/fisiología , Diferenciación Celular/fisiología , Ciclina D1/fisiología , Proteínas de Unión al ADN/metabolismo , Interleucina-6/farmacología , Macrófagos/citología , Macrófagos/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Células Clonales , Ciclina D1/genética , Proteínas de Unión al ADN/genética , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Humanos , Interleucina-4/farmacología , Leucemia Experimental/inmunología , Leucemia Experimental/patología , Leucemia Mieloide/inmunología , Leucemia Mieloide/patología , Macrófagos/efectos de los fármacos , Ratones , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Factores de Transcripción/genética , Transfección , Células Tumorales CultivadasRESUMEN
A new factor-independent megakaryoblastic cell line, designated SET-2, was established from the peripheral blood of a patient with leukemic transformation of essential thrombocythemia (ET). SET-2 expressed CD 4, 7, 13, 33, 34, 36, 38, 41, 61, 71, 117, 126, 130 and c-mpl. In addition, it spontaneously produced numerous platelet-like particles in liquid culture. These particles were shown to be the same size as normal platelets, and to express CD 36, 38, 41, 61 and 71. Proliferation of SET-2 was not influenced by thrombopoietin (TPO) and other hemopoietic cytokines. SET-2 was found to express the platelet-specific proteins such as platelet factor 4 and beta-thromboglobulin. The levels of expression were not altered by TPO. SET-2 also secreted interleukin-6 into the supernatants, as well as normal megakaryocytes. These results suggest that SET-2 spontaneously matures to megakaryocytes and produces platelet-like particles. These findings indicate that SET-2 may be useful for investigating the proliferation and differentiation mechanisms of leukemia cells and the role of c-mpl on megakaryoblasts, megakaryocytes, and platelets in ET. Leukemia (2000) 14, 142-152.
Asunto(s)
Plaquetas/citología , Línea Celular , Megacariocitos/citología , Trombocitopenia/patología , Anciano , Antígenos CD/análisis , División Celular/efectos de los fármacos , Citocinas/farmacología , Femenino , Citometría de Flujo , Humanos , Inmunofenotipificación , Interleucina-6/metabolismo , Megacariocitos/inmunología , Megacariocitos/ultraestructura , Microscopía Electrónica , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trombocitopenia/tratamiento farmacológico , Trombocitopenia/inmunología , Trombopoyetina/genética , Trombopoyetina/metabolismo , Células Tumorales CultivadasRESUMEN
The ubiquitin-proteasome pathway is responsible for selective degradation of short-lived cellular proteins and is critical for the regulation of many cellular processes. We previously showed that ubiquitin (Ub) secreted from hairy cell leukemia cells had inhibitory effects on clonogenic growth of normal hematopoietic progenitor cells. In this study, we examined the effects of exogenous Ub on the growth and survival of a series of human hematopoietic cells, including myeloid cell lines (HL-60 and U937), a B-cell line (Daudi), and T-cell lines (KT-3, MT-4, YTC-3, and MOLT-4). Exogenous Ub inhibited the growth of various hematopoietic cell lines tested, especially of KT-3 and HL-60 cells. The growth-suppressive effects of Ub on KT-3 and HL-60 cells were almost completely abrogated by the proteasome inhibitor PSI or MG132, suggesting the involvement of the proteasome pathway in this process. Furthermore, exogenous Ub evoked severe apoptosis of KT-3 and HL-60 cells through the activation of caspase-3. In interleukin-6 (IL-6)-dependent KT-3 cells, STAT3 was found to be conjugated by exogenous biotinylated Ub and to be degraded in a proteasome-dependent manner, whereas expression levels of STAT1, STAT5, or mitogen-activated protein kinase were not affected. Moreover, IL-6-induced the up-regulation of Bcl-2 and c-myc, and JunB was impaired in Ub-treated KT-3 cells, suggesting that the anti-apoptotic and mitogenic effects of IL-6 were disrupted by Ub. These results suggest that extracellular Ub was incorporated into hematopoietic cells and mediated their growth suppression and apoptosis through proteasome-dependent degradation of selective cellular proteins such as STAT3. (Blood. 2000;95:2577-2585)
Asunto(s)
Apoptosis/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas/patología , Interleucina-6/metabolismo , Complejos Multienzimáticos/metabolismo , Transactivadores/metabolismo , Ubiquitinas/farmacología , Células HL-60 , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal , Factor de Transcripción STAT3 , Transducción de Señal/efectos de los fármacos , Células U937RESUMEN
The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde. We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening. Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated. Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function. We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters.