Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Resistencia a Antineoplásicos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/genética , Pirimidinas/uso terapéuticoAsunto(s)
Antioxidantes/uso terapéutico , Benzamidas/efectos adversos , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/prevención & control , Mutación/genética , Piperazinas/efectos adversos , Pirimidinas/efectos adversos , Vitamina E/uso terapéutico , Animales , Antineoplásicos/efectos adversos , Humanos , Mesilato de Imatinib , Subunidad gamma Común de Receptores de Interleucina/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
RAD51 is one of six mitotic human homologs of the E. coli RecA protein (RAD51-Paralogs) that play a central role in homologous recombination and repair of DNA double-strand breaks (DSBs). Here we demonstrate that RAD51 is important for resistance to cisplatin and mitomycin C in cells expressing the BCR/ABL oncogenic tyrosine kinase. BCR/ABL significantly enhances the expression of RAD51 and several RAD51-Paralogs. RAD51 overexpression is mediated by a STAT5-dependent transcription as well as by inhibition of caspase-3-dependent cleavage. Phosphorylation of the RAD51 Tyr-315 residue by BCR/ABL appears essential for enhanced DSB repair and drug resistance. Induction of the mammalian RecA homologs establishes a unique mechanism for DNA damage resistance in mammalian cells transformed by an oncogenic tyrosine kinase.
Asunto(s)
Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Resistencia a Medicamentos/fisiología , Proteínas de Fusión bcr-abl/metabolismo , Proteínas de la Leche , Rec A Recombinasas/metabolismo , Antibióticos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular , Cisplatino/farmacología , Proteínas de Unión al ADN/genética , Activación Enzimática , Proteínas de Fusión bcr-abl/genética , Genes Reporteros/genética , Humanos , Interleucina-3/farmacología , Microscopía Fluorescente , Mitomicina/farmacología , Fosforilación , Recombinasa Rad51 , Rec A Recombinasas/genética , Factor de Transcripción STAT5 , Transactivadores/genética , Transactivadores/metabolismo , Activación TranscripcionalRESUMEN
The NPM/ALK fusion gene, formed by the t(2;5) translocation in anaplastic large-cell lymphoma, encodes a M(r) 75,000 hybrid protein that containsthe amino-terminal portion of the nucleolar phosphoprotein nucleophosmin(NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocation. Our studies show that NPM/ALK, similar to other members of this family, activates signal transducer and activator of transcription 5 (STAT5) and that this activation is essential for lymphomagenesis. NPM/ALK-mediated activation of STAT5 was demonstrated by detection of: (a) constitutive tyrosine phosphorylation and enhanced DNA binding ability of STAT5 in NPM/ALK-transformed cells; and (b) NPM/ALK-dependent stimulation of STAT5-mediated transactivation of the beta-casein promoter. Retroviral infection of NPM/ALK+ cells with a dominant-negative STAT5B mutant (STAT5-DNM) inhibited the antiapoptotic activity of NPM/ALK in growth factor and serum-free medium. In addition, STAT5-DNM inhibited proliferation and diminished the clonogenic properties of NPM/ALK-positive cells. Finally, SCID mice injected with NPM/ALK+ cells infected with a virus carrying STAT5-DNM survived significantly longer than mice inoculated with NPM/ALK+ cells infected with the empty virus. Necropsy identified a widespread ALK+ lymphoma in lymph nodes and liver of the affected animals. Together, our data indicate that NPM/ALK-induced activation of STAT5 may play an important role in NPM/ALK-mediated lymphomagenesis.
Asunto(s)
Transformación Celular Neoplásica , Proteínas de Unión al ADN/fisiología , Linfocitos/fisiología , Linfoma/patología , Proteínas de la Leche , Proteínas Tirosina Quinasas/fisiología , Transactivadores/fisiología , Animales , Proteínas de Unión al ADN/metabolismo , Femenino , Sustancias de Crecimiento/fisiología , Humanos , Linfocitos/metabolismo , Linfocitos/patología , Linfoma/genética , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Fosforilación , Proteínas Tirosina Quinasas/genética , Factor de Transcripción STAT5 , Transactivadores/metabolismo , TransfecciónRESUMEN
The NPM/ALK fusion gene, formed by the t(2;5) translocation in a subset of anaplastic large cell lymphomas, encodes a Mr 75,000 hybrid protein that contains the NH2-terminal portion of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocations. Our studies showed that NPM/ALK, similar to other members of this family, activates phosphatidylinositol 3-kinase (PI3K) and its downstream effector, serine/threonine kinase (Akt). PI3K was found in complex with NPM/ALK. Both PI3K and Akt kinase were permanently activated in NPM/ALK-transfected BaF3 murine hematopoietic cells and in NPM/ALK-positive, but not in NPM/ALK-negative, patient-derived anaplastic large cell lymphoma cell lines. In addition, Akt was phosphorylated/activated in protein samples isolated from four patients diagnosed with ALK-positive T/null-cell lymphomas. The PI3K inhibitors wortmannin and LY294002 induced apoptosis in NPM/ALK+ cells but exerted only minor effects on the control BaF3 parental cells and peripheral blood mononuclear cells stimulated by growth factors. Furthermore, retroviral infection of NPM/ALK+ BaF3 cells with a dominant-negative PI3K mutant (delta p85) or a dominant-negative Akt mutant (K179M) inhibited proliferation and clonogenic properties of the infected cells. Finally, the Akt mutant (K179M) suppressed the tumorigenicity of NPM/ALK-transfected BaF3 cells injected into syngeneic mice. In conclusion, our data indicate that NPM/ALK constitutively activates the PI3K-Akt pathway and that this pathway plays an important role in the NPM/ALK-mediated malignant transformation.
Asunto(s)
Transformación Celular Neoplásica/patología , Linfoma no Hodgkin/enzimología , Linfoma no Hodgkin/patología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/fisiología , Animales , Línea Celular Transformada , Medios de Cultivo , Activación Enzimática , Femenino , Sustancias de Crecimiento/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Ratones , Ratones Endogámicos BALB C , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-aktRESUMEN
Our previous study indicated that BCR/ABL SH2 domain and BCR/ABL SH3 domain+SH2 domain complex are required for immediate activation of the phosphatidylinositol-3 kinase PI-3k)--> Akt serine/threonine kinase pathway and of the signal transducer and activator of transcription 5 (STAT5), respectively, in hematopoietic cells. We show here that the defect in activation of PI-3k/Akt by BCR/ABL DeltaSH2 mutant (SH2 domain deleted) and of STAT5 by BCR/ABL DeltaSH3+DeltaSH2 mutant (SH3 and SH2 domains deleted) is not permanent and both Akt and STAT5 could be 're-activated' by in vitro culture. This phenomenon was responsible for increased resistance to apoptosis, growth factor-independent proliferation and leukemogenesis in SCID mice. Incubation of cells with BCR/ABL tyrosine kinase inhibitor STI571 abrogated the 're-activation' of Akt or STAT5 by BCR/ABL SH3+SH2 mutants in some clones, in the others Akt and STAT5 activation became independent on BCR/ABL kinase activity. The immediate upstream activators of Akt and STAT5 such as PI-3k and Jak-2 were also activated. In addition, the common beta subunit of IL-3/IL-5/GM-CSF receptor was tyrosine phosphorylated in the clones in which 're-activation' was dependent on the BCR/ABL kinase activity. These results suggested that 're-activation' of Akt and STAT5, in the absence of functional BCR/ABL SH3+SH2 domains, may be achieved by two different mechanisms: (i) BCR/ABL kinase-dependent activation of alternative pathway(s) and (ii) additional genetic changes stimulating Akt and STAT5 independently of BCR/ABL. Oncogene (2000) 19, 4117 - 4124
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Regulación Leucémica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Animales , Apoptosis , Línea Celular Transformada , Proteínas de Fusión bcr-abl/metabolismo , Janus Quinasa 2 , Leucemia Mieloide , Ratones , Ratones SCID , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Pruebas de Precipitina , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Eliminación de Secuencia , Transducción de Señal , Dominios Homologos srcRESUMEN
The Akt serine/threonine kinase is required for the survival of many cell types and for transformation of hematopoietic cells by the BCR/ABL oncogenic tyrosine kinase. Analysis of the potential mechanisms whereby Akt promotes survival of hematopoietic cells revealed that it induced the activity of plasma membrane and mitochondrial Raf-1 in a Ras-independent, but PKC-dependent manner. Inhibition of plasma membrane Raf-1-dependent mitogen-activated protein kinase activity had no effect on the enhanced survival of cells expressing Akt. By contrast, suppression of mitochondrial Raf-1 enzymatic activity by expression of a mitochondria-targeted Raf-1 dominant-negative mutant rendered Akt-expressing cells susceptible to apoptosis induced by growth factor deprivation and was accompanied by inhibition of BAD, but not mitogen-activated protein kinase, phosphorylation. Together, these data indicate that PKC-dependent activation of Raf-1 plays an important role in Akt-dependent antiapoptotic effects.
Asunto(s)
Apoptosis/fisiología , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Oncogénicas de Retroviridae/fisiología , Animales , Línea Celular , Activación Enzimática , Ratones , Proteína Oncogénica v-aktRESUMEN
Signal transducer and activator of transcription (STAT)5 is constitutively activated in BCR/ ABL-expressing cells, but the mechanisms and functional consequences of such activation are unknown. We show here that BCR/ABL induces phosphorylation and activation of STAT5 by a mechanism that requires the BCR/ABL Src homology (SH)2 domain and the proline-rich binding site of the SH3 domain. Upon expression in 32Dcl3 growth factor-dependent myeloid precursor cells, STAT5 activation-deficient BCR/ABL SH3+SH2 domain mutants functioned as tyrosine kinase and activated Ras, but failed to protect from apoptosis induced by withdrawal of interleukin 3 and/or serum and did not induce leukemia in severe combined immunodeficiency mice. In complementation assays, expression of a dominant-active STAT5B mutant (STAT5B-DAM), but not wild-type STAT5B (STAT5B-WT), in 32Dcl3 cells transfected with STAT5 activation-deficient BCR/ABL SH3+SH2 mutants restored protection from apoptosis, stimulated growth factor-independent cell cycle progression, and rescued the leukemogenic potential in mice. Moreover, expression of a dominant-negative STAT5B mutant (STAT5B-DNM) in 32Dcl3 cells transfected with wild-type BCR/ABL inhibited apoptosis resistance, growth factor-independent proliferation, and the leukemogenic potential of these cells. In retrovirally infected mouse bone marrow cells, expression of STAT5B-DNM inhibited BCR/ABL-dependent transformation. Moreover, STAT5B-DAM, but not STAT5B-WT, markedly enhanced the ability of STAT5 activation-defective BCR/ABL SH3+SH2 mutants to induce growth factor-independent colony formation of primary mouse bone marrow progenitor cells. However, STAT5B-DAM did not rescue the growth factor-independent colony formation of kinase-deficient K1172R BCR/ABL or the triple mutant Y177F+R522L+ Y793F BCR/ABL, both of which also fail to activate STAT5. Together, these data demonstrate that STAT5 activation by BCR/ABL is dependent on signaling from more than one domain and document the important role of STAT5-regulated pathways in BCR/ABL leukemogenesis.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes abl/genética , Leucemia/genética , Proteínas de la Leche , Transactivadores/genética , Dominios Homologos src/genética , Animales , Apoptosis , Células de la Médula Ósea/metabolismo , Ciclo Celular/genética , Replicación del ADN/genética , Genes ras/genética , Ratones , Ratones SCID , Mutación , Fosfoproteínas/análisis , Fosforilación , Factor de Transcripción STAT5 , Transducción de Señal/genética , Células Madre/metabolismo , Activación Transcripcional/genéticaRESUMEN
The phenotype of hematopoietic cells transformed by the BCR/ABL oncoprotein of the Philadelphia chromosome is characterized by growth factor-independent proliferation, reduced susceptibility to apoptosis, and altered adhesion and motility. The mechanisms underlying this phenotype are not fully understood, but there is evidence that some of the properties of BCR/ABL-expressing cells are dependent on the activation of downstream effector molecules such as RAS, PI-3k, and bcl-2. We show here that the small GTP-binding protein Rac is activated by BCR/ABL in a tyrosine kinase-dependent manner. Upon transfection with a vector carrying the dominant-negative N17Rac, BCR/ABL-expressing myeloid precursor 32Dcl3 cells retained the resistance to growth factor deprivation-induced apoptosis but showed a decrease in proliferative potential in the absence of interleukin-3 (IL-3) and markedly reduced invasive properties. Moreover, compared with BCR/ABL-expressing cells, fewer BCR/ABL plus N17Rac double transfectants were capable of homing to bone marrow and spleen. Consistent with these findings, survival of SCID mice injected with the BCR/ABL plus N17Rac double transfectants was markedly prolonged as compared with that of mice injected with BCR/ABL-expressing cells. Together, these data support the important role of a Rac-dependent pathway(s) controlling motility in BCR/ABL-mediated leukemogenesis.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Proteínas de Unión al GTP/metabolismo , Leucemia Experimental/etiología , Leucemia Experimental/genética , Animales , Secuencia de Bases , División Celular , Línea Celular Transformada , Movimiento Celular , Supervivencia Celular , Cartilla de ADN/genética , Proteínas de Fusión bcr-abl/metabolismo , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Experimental/metabolismo , Ratones , Ratones SCID , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Fenotipo , Proteínas de Unión al GTP racRESUMEN
To determine the possible role of the BCR/ABL oncoprotein SH3 domain in BCR/ABL-dependent leukemogenesis, we studied the biologic properties of a BCR/ABL SH3 deletion mutant (delta SH3 BCR/ABL) constitutively expressed in murine hematopoietic cells. delta SH3 BCR/ABL was able to activate known BCR/ABL-dependent downstream effector molecules such as RAS, PI-3kinase, MAPK, JNK, MYC, JUN, STATs, and BCL-2. Moreover, expression of delta SH3 BCR/ABL protected 32Dcl3 murine myeloid precursor cells from apoptosis, induced their growth factor-independent proliferation, and resulted in transformation of primary bone marrow cells in vitro. Unexpectedly, leukemic growth from cells expressing delta SH3 BCR/ABL was significantly retarded in SCID mice compared with that of cells expressing the wild-type protein. In vitro and in vivo studies to determine the adhesive and invasive properties of delta SH3 BCR/ABL-expressing cells showed their decreased interaction to collagen IV- and laminin-coated plates and their reduced capacity to invade the stroma and to seed the bone marrow and spleen. The decreased interaction with collagen type IV and laminin was consistent with a reduced expression of alpha 2 integrin by delta SH3 BCR/ABL-transfected 32Dcl3 cells. Moreover, as compared with wild-type BCR/ABL, which localizes primarily in the cytoskeletal/membrane fraction, delta SH3 BCR/ABL was more evenly distributed between the cytoskeleton/membrane and the cytosol compartments. Together, the data indicate that the SH3 domain of BCR/ABL is dispensable for in vitro transformation of hematopoietic cells but is essential for full leukemogenic potential in vivo.
Asunto(s)
Movimiento Celular/genética , Transformación Celular Neoplásica , Proteínas de Fusión bcr-abl/genética , Regulación Neoplásica de la Expresión Génica , Leucemia Experimental/patología , Dominios Homologos src/genética , Animales , Adhesión Celular/genética , Línea Celular , Leucemia Experimental/genética , RatonesRESUMEN
The BCR/ABL oncogenic tyrosine kinase activates phosphatidylinositol 3-kinase (PI-3k) by a mechanism that requires binding of BCR/ABL to p85, the regulatory subunit of PI-3k, and an intact BCR/ABL SH2 domain. SH2 domain BCR/ABL mutants deficient in PI-3k activation failed to stimulate Akt kinase, a recently identified PI-3k downstream effector with oncogenic potential, but did activate p21 RAS and p70 S6 kinase. The PI-3k/Akt pathway is essential for BCR/ABL leukemogenesis as indicated by experiments demonstrating that wortmannin, a PI-3k specific inhibitor at low concentrations, suppressed BCR/ABL-dependent colony formation of murine marrow cells, and that a kinase-deficient Akt mutant with dominant-negative activity inhibited BCR/ABL-dependent transformation of murine bone marrow cells in vitro and suppressed leukemia development in SCID mice. In complementation assays using mouse marrow progenitor cells, the ability of transformation-defective SH2 domain BCR/ABL mutants to induce growth factor-independent colony formation and leukemia in SCID mice was markedly enhanced by expression of constitutively active Akt. In retrovirally infected mouse marrow cells, the BCR/ABL mutant lacking the SH2 domain was unable to upregulate the expression of c-Myc and Bcl-2; in contrast, expression of a constitutively active Akt mutant induced Bcl-2 and c-Myc expression, and stimulated the transcription activation function of c-Myc. Together, these data demonstrate the requirement for the BCR/ABL SH2 domain in PI-3k activation and document the essential role of the PI-3k/Akt pathway in BCR/ABL leukemogenesis.
Asunto(s)
Células de la Médula Ósea , Transformación Celular Neoplásica/genética , Proteínas de Fusión bcr-abl/genética , Leucemia Experimental/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Animales , Médula Ósea/patología , Activación Enzimática , Genes bcl-2 , Genes myc , Leucemia Experimental/etiología , Leucemia Experimental/patología , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Bazo/patologíaRESUMEN
We studied the effect of phosphorothioate oligodeoxynucleotides ([S]ODNs) complementary to the bcr-abl junction on cells taken at diagnosis from 41 patients with Philadelphia-positive chronic myelogenous leukaemia (CML). Experiments included the evaluation of the anti-leukaemic effect of 16- and 26-mer antisense [S]ODNs on both mononuclear and CD34+ cells, evaluation of incubation time and correlation of colony growth inhibition with the down-regulation of p210(bcr-abl). At the same time, the uptake of [S]ODNs by mononuclear and purified CD34+ cell populations and the cross-hybridization of 26- and 16-mer [S]ODNs with the complementary sequences were evaluated. After incubation for 120 h with 26-mer antisense [S]ODNs on mononuclear cells, overall mean colony recovery was 41.9% of the untreated control samples; in particular, a significant reduction in colony formation was observed in 22 of the 35 cases tested. The effect of 26-mer ODNs on CD34+ cells was comparable to that observed on mononuclear cells in terms of colony inhibition; however, a higher proportion of cases showed a significant inhibition of colony formation. In comparison with the 26-mer antisense [S]ODNs, the anti-leukaemic effect of the 16-mer antisense [S]ODNs was less evident on mononuclear cells and comparable on CD34+ cells; however, a more specific effect was evident on both target cells. Hybridization experiments confirmed a partial cross-reactivity when the 26-mer ODNs were hybridized with their complementary sequence; this did not occur when 16-mer ODNs were similarly tested. Experiments aimed at evaluating the effect of the incubation time showed a significant increase in anti-leukaemic effect after a 120 h incubation period compared to that measured after a 24 h incubation period; this was parallelled by a progressive increase in the intracellular concentrations of [S]ODNs from day 1 to day 5. The accumulation of [S]ODNs correlated with a marked down-regulation of p210(bcr-abl) levels which was first detectable after 72 h of treatment. The down-regulation of p210(bcr-abl) levels following treatment with [S]ODNs showed a correlation between the effect of antisense [S]ODNs on leukaemic colony formation and protein expression. These studies confirm that, under optimal conditions of target cell culture and ODN size, antisense [S]ODNs complementary to the bcr-abl junction have specific anti-leukaemic effects.
Asunto(s)
División Celular/efectos de los fármacos , Proteínas de Fusión bcr-abl/genética , Células Madre Hematopoyéticas/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Oligonucleótidos Antisentido/toxicidad , Antígenos CD/análisis , Antígenos CD34/análisis , Secuencia de Bases , Transporte Biológico , Médula Ósea/patología , Células de la Médula Ósea , Células Clonales , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/patología , Humanos , Cinética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/patología , Oligonucleótidos Antisentido/farmacocinética , Reacción en Cadena de la Polimerasa , Tionucleótidos , Células Tumorales CultivadasRESUMEN
In vitro, uniformly modified oligonucleotide N3'-->P5' phosphoramidates are apparently more potent antisense agents than phosphorothioate derivatives. To determine whether such compounds are also effective in vivo, severe combined immunodeficiency mice injected with HL-60 myeloid leukemia cells were treated systemically with equal doses of either phosphoramidate or phosphorothioate c-myc antisense or mismatched oligonucleotides. Compared with mice treated with mismatched oligodeoxynucleotides, the peripheral blood leukemic load of mice treated with the antisense sequences was markedly reduced, and such effects were associated with significantly prolonged survival of the antisense-treated mice. Moreover, with each of three different treatment schedules (100, 300, or 900 microg/day for 6 consecutive days), survival of the phosphoramidate-treated mice was significantly longer than that of the phosphorothioate-treated mice. Both phosphoramidate and phosphorothioate oligonucleotides were efficiently taken up by leukemic cells in vivo and were capable of specifically down-regulating c-Myc expression. Moreover, tissue distribution of the phosphoramidate derivatives was undistinguishable from that of the phosphorothioate derivatives. Collectively, these studies suggest that phosphoramidate oligonucleotides can serve as potent and specific antisense agents in the treatment of human leukemia and probably of other malignancies.
Asunto(s)
Genes myc , Leucemia Experimental/terapia , Oligonucleótidos Antisentido/uso terapéutico , Animales , Humanos , Leucemia Experimental/genética , Masculino , Ratones , Ratones Endogámicos ICR , Ratones SCIDRESUMEN
BACKGROUND: Philadelphia cells are human chronic myelogenous leukemia (CML) cells that contain the BCR/ABL oncogene (a fusion of the BCR and ABL genes). Selective eradication of these cells in vitro can be achieved by combined treatment with antisense phosphorothioate oligodeoxynucleotides ([S]ODNs) specifically targeted to this oncogene (bcr/abl [S]ODNs) and a suboptimal (for use as a single agent) dose of mafosfamide (the in vitro active form of cyclophosphamide). PURPOSE: We evaluated the ability of bcr/abl antisense [S]ODNs, alone or subsequent to treatment with a single injection of cyclophosphamide, to suppress the leukemic process induced in severe combined immunodeficient (SCID) mice by Philadelphia cells (i.e., primary CML-blast crisis [CML-BC] cells). In addition, we studied potential mechanisms that might explain the efficacy of the bcr/abl antisense [S]ODN-mafosfamide combination against Philadelphia cells in vitro. METHODS: The effects of treating leukemic mice with cyclophosphamide (25 mg/kg body weight; 25% of the dose required to eradicate evidence of leukemia in SCID mice) and/or bcr/abl antisense [S]ODNs were assessed by analysis of survival, by examination of bone marrow for the presence of leukemia cells (using a colony formation assay or using coupled reverse transcription and the polymerase chain reaction to screen for bcr/abl messenger RNA), and by examination of a variety of tissues for the presence of infiltrating leukemia cells. The induction of apoptosis (a cell death program) in vitro in primary CML-BC cells following treatment with bcr/abl antisense [S]ODNs plus or minus prior treatment with mafosfamide was monitored by use of a commercial assay. Relative cellular uptake of [S]ODNs by CML-BC cells treated in vitro with or without prior treatment with mafosfamide was determined by use of confocal microscopy and flow cytometry (for fluorescent [S]ODNs) or by use of blotting techniques that employed radioactively labeled probes (for extracted, unlabeled [S]ODNs). Levels of specific proteins in treated and untreated cells were determined by use of western blotting methods. Reported P values are two-sided. RESULTS: The disease process in leukemic mice was retarded substantially by combination treatment with cyclophosphamide and specific bcr/abl antisense [S]ODNs (P < .001, relative to treatment with specific antisense [S]ODNs alone, cyclophosphamide alone, or cyclophosphamide plus nonspecific [i.e., control] antisense [S]ODNs); 50% of the mice treated with cyclophosphamide and specific antisense [S]ODNs appeared to be cured of leukemia. The combination treatment was associated with increased induction of apoptosis. In addition, cellular uptake of bcr/abl antisense [S]ODNs appeared to be increased twofold to sixfold by prior treatment with mafosfamide. This increased uptake of [S]ODNs was associated with enhanced suppression of p210bcr/abl protein levels. CONCLUSIONS AND IMPLICATIONS: Combination therapy with antisense [S]ODNs targeted to specific oncogenes and less toxic doses of anticancer drugs may represent a rational strategy to purpose for the treatment of human leukemias.
Asunto(s)
Antineoplásicos/uso terapéutico , Ciclofosfamida/análogos & derivados , Proteínas de Fusión bcr-abl/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Cromosoma Filadelfia , Tionucleótidos/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , Ciclofosfamida/uso terapéutico , Sondas de ADN , Citometría de Flujo , Proteínas de Fusión bcr-abl/biosíntesis , Proteínas de Fusión bcr-abl/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Ratones , Ratones SCID , Reacción en Cadena de la Polimerasa , Análisis de Supervivencia , Resultado del Tratamiento , Células Tumorales CultivadasRESUMEN
Blastic transformation of chronic myelogenous leukemia (CML) is characterized by the presence of nonrandom, secondary genetic abnormalities in the majority of Philadelphia1 clones, and loss of p53 tumor suppressor gene function is a consistent finding in 25-30% of CML blast crisis patients. To test whether the functional loss of p53 plays a direct role in the transition of chronic phase to blast crisis, bone marrow cells from p53+/+ or p53-/- mice were infected with a retrovirus carrying either the wild-type BCR/ABL or the inactive kinase-deficient mutant, and were assessed for colony-forming ability. Infection of p53-/- marrow cells with wild-type BCR/ABL, but not with the kinase-deficient mutant, enhanced formation of hematopoietic colonies and induced growth factor independence at high frequency, as compared with p53+/+ marrow cells. These effects were suppressed when p53-/- marrow cells were coinfected with BCR/ ABL and wild-type p53. p53-deficient BCR/ABL-infected marrow cells had a proliferative advantage, as reflected by an increase in the fraction of S+G2 phase cells and a decrease in the number of apoptotic cells. Immunophenotyping and morphological analysis revealed that BCR/ABL-positive p53-/- cells were much less differentiated than their BCR/ABL-positive p53+/+ counterparts. Injection of immunodeficient mice with BCR/ABL-positive p53-/- cells produced a transplantable, highly aggressive, poorly differentiated acute myelogenous leukemia. In marked contrast, the disease process in mice injected with BCR/ABL-positive p53+/+ marrow cells was characterized by cell infiltrates with a more differentiated phenotype and was significantly retarded, as indicated by a much longer survival of leukemic mice. Together, these findings directly demonstrate that loss of p53 function plays an important role in blast transformation in CML.
Asunto(s)
Crisis Blástica , Médula Ósea/patología , Proteínas de Fusión bcr-abl/metabolismo , Genes p53 , Interleucina-3/farmacología , Interleucina-6/farmacología , Leucemia Mieloide Aguda/patología , Proteínas Tirosina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Animales , Antígenos CD34/análisis , Apoptosis/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , Complejo CD3/análisis , Ciclo Celular/efectos de los fármacos , Proteínas de Fusión bcr-abl/biosíntesis , Expresión Génica , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/genética , Antígenos Comunes de Leucocito/análisis , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Recombinantes/farmacología , Retroviridae , Bazo/inmunología , Bazo/patologíaRESUMEN
The proliferation of chronic myelogenous leukemia (CML) cells and the transformation of normal hematopoietic cells by BCR-ABL appear to require the expression of a functional MYC protein, suggesting an approach to treatment of Philadelphia leukemias based on simultaneous targeting of BCR-ABL and c-MYC. To test this hypothesis, CML-blast crisis (CML-BC) primary cells were treated in vitro with bcr-abl and c-myc antisense phosphorothioate oligodeoxynucleotides ([S]ODNs), individually or in combination. Compared with antisense ODNs targeting of individual oncogenes, downregulation of both BCR-ABL and c-MYC by specific antisense [S]ODNs resulted in a synergistic antiproliferative effect. Colony formation of normal bone marrow cells was not affected by either treatment. To assess the therapeutic potential of multiple oncogene downregulation, SCID mice injected with CML-BC primary cells were treated systematically with equal doses of bcr-abl or c-myc antisense [S]ODNs or with a combination of both antisense [S]ODNs. Compared with mice treated with individual compounds, the disease process was significantly retarded in the group treated with both [S]ODNs as revealed by flow cytometry, clonogenic assay, and RT-PCR analysis to detect leukemic cells in mouse tissue cell suspensions. These effects correlated with a markedly increased survival of leukemic mice treated with both antisense [S]ODNs. Leukemic cells harvested from antisense [S]ODN-treated mice were sensitive to the effects of antisense [S]ODNs in vitro, suggesting that the treatment can be successfully repeated. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.
Asunto(s)
Crisis Blástica/terapia , Proteínas de Fusión bcr-abl/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/efectos de los fármacos , Oligonucleótidos Antisentido/uso terapéutico , Proteínas Proto-Oncogénicas c-myc/genética , Tionucleótidos/uso terapéutico , Animales , Secuencia de Bases , Crisis Blástica/genética , División Celular/efectos de los fármacos , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Ratones SCID , Datos de Secuencia Molecular , Trasplante de Neoplasias , Ensayo de Tumor de Célula MadreRESUMEN
Uniformly modified oligonucleotide N3'-->P5' phosphoramidates, where every 3'-oxygen is replaced by a 3'-amino group, were synthesized. These compounds have very high affinity to single-stranded RNAs and thus have potential utility as antisense agents. As was shown in this study, the oligonucleotide phosphoramidates are resistant to digestion with snake venom phosphodiesterase, to nuclease activity in a HeLa cell nuclear extract, or to nuclease activity in 50% human plasma, where no significant hydrolysis was observed after 8 h. These compounds were used in various in vitro cellular systems as antisense compounds addressed to different targeted regions of c-myb, c-myc and bcr-abl mRNAs. C-myb antisense phosphoramidates at 5 microM caused sequence and dose-dependent inhibition of HL-60 cell proliferation and a 75% reduction in c-myb protein and RNA levels, as determined by Western blot and RT-PCR analysis. Analogous results were observed for anti-c-myc phosphoramidates, where a complete cytostatic effect for HL-60 cells was observed at 1 microM concentration for fully complementary, but not for mismatched compounds, which were indistinguishable from untreated controls. This was correlated with a 93% reduction in c-myc protein level. Moreover, colony formation by the primary CML cells was also inhibited 75-95% and up to 99% by anti-c-myc and anti-bcr-abl phosphoramidate oligonucleotides, respectively, in a sequence- and dose-dependent manner within a 0.5 nM-5 microM dose range. At these concentrations the colony-forming ability of normal bone marrow cells was not affected. The presented in vitro data indicate that oligonucleotide N3'-->P5' phosphoramidates could be used as specific and efficient antisense agents.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Proteínas Nucleares/metabolismo , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Animales , Secuencia de Bases , Western Blotting , División Celular/efectos de los fármacos , Línea Celular , Electroforesis en Gel de Poliacrilamida , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Hidrólisis , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , Proteínas Proto-Oncogénicas c-myb , Células Tumorales CultivadasRESUMEN
To characterize the distribution and toxicity of phosphorothioate antisense oligodeoxynucleotides ([S]ODNs) in vivo, the mice, previously injected with BV173 leukemic cells (Philadelphia chromosome-positive chronic myeloid leukemia blast-crisis), received intravenously 26-mer BCR-ABL antisense oligodeoxynucleotides (1 mg/mouse/day) for 9 consecutive days. Our investigation revealed that [S]ODNs were distributed to almost all organs except the brain with the highest level in the liver, spleen and kidneys. They were also detected in CD10+ leukemic cells isolated from spleen and bone marrow. Intracellular distribution assay showed the presence of [S]ODNs most prominently in nuclear and cytoplasmic fractions. Our data demonstrated no significant toxicity of [S]ODNs except the increase in spleen weight.
Asunto(s)
Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/farmacocinética , Tionucleótidos/farmacología , Tionucleótidos/farmacocinética , Animales , Crisis Blástica/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Transformation of hematopoietic cells by the p210bcr/abl tyrosine kinase appears to require the expression of a functional MYC protein, suggesting that simultaneous targeting of BCR-ABL and c-myc might be a rational strategy for attempting treatment of Phil-adelphia leukemia. To test this hypothesis, severe combined immunodeficiency mice injected with Philadelphia leukemic cells were treated systemically with equal doses of bcr-abl or c-myc antisense oligodeoxynucleotides (ODNs) or with both ODNs in combination. Compared with the mice treated with individual agents, the disease process was much slower in the group treated with both ODNs, as revealed by flow cytometry, clonogenic assay, and reverse transcriptase-polymerase chain reaction analysis to detect leukemic cells in mouse tissue cell suspensions, and by enumeration of liver metastases. The retardation of the disease process was positively correlated with a markedly increased survival of leukemic mice treated with both ODNs. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Genes myc , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Animales , Secuencia de Bases , Cartilla de ADN/química , Expresión Génica , Masculino , Ratones , Ratones SCID , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Neprilisina/análisis , Oncogenes , ARN Mensajero/genética , ARN Neoplásico/genética , Células Tumorales CultivadasRESUMEN
The BCR/ABL oncogenic tyrosine kinase is responsible for initiating and maintaining the leukemic phenotype of Philadelphia chromosome (Ph1)-positive cells. Phosphatidylinositol-3 (PI-3) kinase is known to interact with and be activated by receptor and nonreceptor tyrosine kinases. We investigated whether PI-3 kinase associates with and/or is regulated by BCR/ABL, whether this interaction is functionally significant for Ph1 cell proliferation, and, if so, whether inhibition of PI-3 kinase activity can be exploited to eliminate Ph1-positive cells from bone marrow. We show that the p85 alpha subunit of PI-3 kinase associates with BCR/ABL and that transient expression of BCR/ABL in fibroblasts and down-regulation of BCR/ABL expression using antisense oligodeoxynucleotides (ODNs) in Ph1 cells activates and inhibits, respectively, PI-3 kinase enzymatic activity. The use of specific ODNs or antisense constructs to downregulate p85 alpha expression showed a requirement for p85 alpha subunit in the proliferation of BCR/ABL-dependent cell lines and chronic myelogenous leukemia (CML) primary cells. Similarly, wortmannin, a specific inhibitor of the enzymatic activity of the p110 subunit of PI-3 kinase, inhibited growth of these cells. The growth of normal bone marrow and erythromyeloid, but not megakaryocyte, progenitors was inhibited by p85 alpha antisense [S]ODNs, but wortmannin, at the concentrations tested, did not affect normal hematopoiesis. The proliferation of two BCR/ABL- and growth factor-independent cell lines was not affected by downregulation of the expression of the p85 alpha subunit or inhibition of p110 enzymatic activity, confirming the specificity of the observed effects on Ph1 cells. Thus, PI-3 kinase is one of the downstream effectors of BCR/ABL tyrosine kinase in CML cells. Moreover, reverse transcriptase-polymerase chain reaction performed on single colonies to detect BCR-ABL transcripts showed that wortmannin was able to eliminate selectively CML-blast crisis cells from a mixture of normal bone marrow and Ph1 cells.