RESUMEN
Telomeres interact with numerous proteins, including components of the shelterin complex, whose alteration, similarly to proliferation-induced telomere shortening, initiates cellular senescence. In tumors, telomere length is maintained by Telomerase activity or by the Alternative Lengthening of Telomeres mechanism, whose hallmark is the telomeric localization of the promyelocytic leukemia (PML) protein. Whether PML contributes to telomeres maintenance in normal cells is unknown. We show that in normal human fibroblasts the PML protein associates with few telomeres, preferentially when they are damaged. Proliferation-induced telomere attrition or their damage due to alteration of the shelterin complex enhances the telomeric localization of PML, which is increased in human T-lymphocytes derived from patients genetically deficient in telomerase. In normal fibroblasts, PML depletion induces telomere damage, nuclear and chromosomal abnormalities, and senescence. Expression of the leukemia protein PML/RARα in hematopoietic progenitors displaces PML from telomeres and induces telomere shortening in the bone marrow of pre-leukemic mice. Our work provides a novel view of the physiologic function of PML, which participates in telomeres surveillance in normal cells. Our data further imply that a diminished PML function may contribute to cell senescence, genomic instability, and tumorigenesis.
Asunto(s)
Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Receptores de Ácido Retinoico/genética , Telómero/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Animales , Carcinogénesis/genética , Línea Celular , Proliferación Celular/genética , Senescencia Celular/genética , Inestabilidad Genómica , Humanos , Ratones , Proteína de la Leucemia Promielocítica , Receptor alfa de Ácido Retinoico , Linfocitos T/patología , Telomerasa/genéticaRESUMEN
MicroRNAs (miRNAs) regulate cell proliferation, differentiation and death during development and postnatal life. The expression level of mature miRNAs results from complex molecular mechanisms, including the transcriptional regulation of their genes. MiR-223 is a hematopoietic-specific miRNA participating in regulatory signaling networks involving lineage-specific transcription factors (TFs). However, the transcriptional mechanisms governing its expression levels and its functional role in lineage fate decision of human hematopoietic progenitors (HPCs) have not yet been clarified. We found that in CD34(+)HPCs undergoing unilineage differentiation/maturation, miR-223 is upregulated more than 10-fold during granulopoiesis, 3-fold during monocytopoiesis and maintained at low levels during erythropoiesis. Chromatin immunoprecipitation and promoter luciferase assays showed that the lineage-specific expression level of mature miR-223 is controlled by the coordinated binding of TFs to their DNA-responsive elements located in 'distal' and 'proximal' regulatory regions of the miR-223 gene, differentially regulating the transcription of two primary transcripts (pri-miRs). All this drives myeloid progenitor maturation into specific lineages. Accordingly, modulation of miR-223 activity in CD34(+)HPCs and myeloid cell lines significantly affects their differentiation/maturation into erythroid, granulocytic and monocytic/macrophagic lineages. MiR-223 overexpression increases granulopoiesis and impairs erythroid and monocytic/macrophagic differentiation. Its knockdown, meanwhile, impairs granulopoiesis and facilitates erythropoiesis and monocytic/macrophagic differentiation. Overall, our data reveal that transcriptional pathways acting on the differential regulation of two pri-miR transcripts results in the fine-tuning of a single mature miRNA expression level, which dictates the lineage fate decision of hematopoietic myeloid progenitors.
Asunto(s)
Linaje de la Célula/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Transcripción Genética/genética , Activación Transcripcional , Antígenos CD34/metabolismo , HumanosAsunto(s)
Proteínas de Unión al ADN/genética , Leucemia Promielocítica Aguda/genética , Mielopoyesis/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Transcripción Genética , Proteínas Supresoras de Tumor/genética , Secuencia de Bases , Línea Celular Tumoral , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/metabolismo , Regiones Promotoras Genéticas , Tretinoina/farmacologíaRESUMEN
The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RAR alpha fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RAR alpha mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RAR alpha alters the early hematopoietic program through N-CoR-dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells. (Blood. 2000;96:1531-1537)
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Leucopoyesis/genética , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Enfermedad Aguda , Diferenciación Celular/genética , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Leucemia Mieloide/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Fusión Oncogénica/biosíntesisRESUMEN
Retinoic Acid (RA) treatment induces disease remission of Acute Promyelocytic Leukaemia (APL) patients by triggering terminal differentiation of neoplastic cells. RA-sensitivity in APL is mediated by its oncogenic protein, which results from the recombination of the PML and the RA receptor alpha (RAR alpha) genes (PML/RAR alpha fusion protein). Ectopic expression of PML/RAR alpha into haemopoietic cell lines results in increased response to RA-induced differentiation. By structure-function analysis of PML/RAR alpha-mediated RA-differentiation, we demonstrated that fusion of PML and RAR alpha sequences and integrity of the PML dimerization domain and of the RAR alpha DNA binding region are required for the effect of PML/RAR alpha on RA-differentiation. Indeed, direct fusion of the PML dimerization domain to the N- or C-terminal extremities of RAR alpha retained full biological activity. All the biologically active PML/RAR alpha mutants formed high molecular weight complexes in vivo. Functional analysis of mutations within the PML dimerization domain revealed that the capacity to form PML/RAR alpha homodimers, but not PML/RAR alpha-PML heterodimers, correlated with the RA-response. These results suggest that targeting of RAR alpha sequences by the PML dimerization domain and formation of nuclear PML/RAR alpha homodimeric complexes are crucial for the ability of PML/RAR alpha to mediate RA-response.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/química , Proteínas de Fusión Oncogénica/química , Tretinoina/farmacología , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Dimerización , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Peso Molecular , Proteínas de Neoplasias/fisiología , Proteínas de Fusión Oncogénica/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Células U937/efectos de los fármacos , Células U937/metabolismo , Dedos de ZincRESUMEN
We analyzed the relationship between the reinfusion of large or very large amounts of peripheral blood progenitor cells (PBPC) and hematologic toxicity in twenty-one advanced breast cancer patients subjected to a myeloablative dose of melphalan at the end of a high-dose sequential chemotherapy (HDSC) program. We also evaluated the influence of the white blood cell (WBC) count to predict an optimal PBPC harvest after high-dose chemotherapy and growth factor priming. Twenty-one patients with high-risk or metastatic breast cancer sequentially received: high-dose cyclophosphamide (HD-Cy) and G-CSF followed by PBPC harvest, HD-methotrexate plus vincristine, HD-doxorubicin, cisplatin and finally HD-melphalan 200 mg/m2 (HD-L-PAM) followed by PBPC reinfusion. No growth factor was administered after HD-L-PAM. CD34+ cytofluorimetric analysis, WBC count and clonogenic assays were employed to monitor circulating cells and to analyze the PBPC harvest. Correlation between different PBPC doses and hematologic toxicity as well as leukocyte and platelet recovery time was attempted. Patients received a median number of 16 (4-25.1) x 10(6)/kg CD34+ cells, 81.3 (30.8-228) x 10(4)/kg CFU-GM and 4.2 (1.3-7.3) x 10(8)/kg nucleated cells (NC) after HD-L-PAM. The number of days with fewer than 1 x 10(9)/l leukocytes and 20 x 10(9)/l platelets were 6 (range 4-9) and 0 (range 0-3), respectively. The CD34+ cell dose significantly correlated with both platelet count nadir (r = 0.73) and time to 50 x 10(9)/l platelets (r = 0.7), but did not correlate with time to reach more than 1 x 10(9)/l WBC count (r = 0.2). In particular, we found that in 12 patients given very large amounts of CD34+ cells, ranging between 15.8 and 25. 1 x 10(6)/kg (V-LA-CD34+), the platelet nadir count never fell below 20 x 10(9)/l and platelet transfusions were not required. Conversely, nine patients who received only large amounts of CD34+ cells, ranging between 4 and 12 x 10(6)/kg (LA-CD34+), experienced a platelet nadir lower than 20 x 10(9)/l and required 2 days (range 1-4) to achieve independence from platelet transfusions (P = 0.001 and P = 0. 0005). The requirement for packed red blood cells (RBC) was 1.5 vs 3 units in the V-LA-CD34+ and LA-CD34+ groups respectively (P = 0.063). The analysis of 44 PBPC collections demonstrated that 29 aphereses performed with a WBC count <20 x 10(9)/l yielded a mean of 312 +/- 43 x 10(6) CD34+ cells and 1831 +/- 201 x 10(4) CFU-GM, whereas 15 collections performed with WBC count >20 x 10(9)/l yielded 553 +/- 64 x 10(6) CD34+ cells and 3190 +/- 432 x 10(4) CFU-GM (P = 0.004). In conclusion, our data suggests that V-LA-CD34+ eliminates severe thrombocytopenia and platelet transfusion requirements in breast cancer patients subjected to HD-L-PAM, and higher PBPC collections seems to coincide with WBC count higher than 20 x 10(9)/l after HD-Cy and G-CSF mobilization. These results justify a prospective study to establish whether large doses of CD34+ cells result in significant clinical benefits.
Asunto(s)
Antineoplásicos Alquilantes/efectos adversos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/terapia , Trasplante de Células Madre Hematopoyéticas , Melfalán/efectos adversos , Trombocitopenia/terapia , Adulto , Antineoplásicos Alquilantes/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/sangre , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Hematopoyesis , Movilización de Célula Madre Hematopoyética , Humanos , Recuento de Leucocitos , Melfalán/administración & dosificación , Persona de Mediana Edad , Recuento de Plaquetas , Trombocitopenia/sangre , Trombocitopenia/inducido químicamente , Trasplante AutólogoRESUMEN
The production of red blood cells follows the sequential formation of proerythroblasts and basophilic, polychromatophilic and orthochromatic erythroblasts, and is promoted by the hormone erythropoietin (Epo) in response to tissue hypoxia. However, little is known about the negative regulation of this process. Death receptors are a family of surface molecules that trigger caspase activation and apoptosis in a variety of cell types. Here we show that immature erythroid cells express several death receptors whose ligands are produced by mature erythroblasts. Exposure of erythroid progenitors to mature erythroblasts or death-receptor ligands resulted in caspase-mediated degradation of the transcription factor GATA-1, which is associated with impaired erythroblast development. Expression of a caspase-resistant GATA-1 mutant, but not of the wild-type gene, completely restored erythroid expansion and differentiation following the triggering of death receptors, indicating that there is regulatory feedback between mature and immature erythroblasts through caspase-mediated cleavage of GATA-1. Similarly, erythropoiesis blockade following Epo deprivation was largely prevented by the expression of caspase-inhibitory proteins or caspase-resistant GATA-1 in erythroid progenitors. Caspase-mediated cleavage of GATA-1 may therefore represent an important negative control mechanism in erythropoiesis.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas de Unión al ADN/fisiología , Eritropoyesis/fisiología , Factores de Transcripción/fisiología , Receptor fas/fisiología , Células Cultivadas , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Eritroblastos/fisiología , Factores de Unión al ADN Específico de las Células Eritroides , Eritropoyetina/fisiología , Proteína Ligando Fas , Factor de Transcripción GATA1 , Factor de Transcripción GATA2 , Humanos , Glicoproteínas de Membrana/fisiología , Mutagénesis , Mutación , Receptores de Superficie Celular/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Receptor fas/inmunologíaRESUMEN
Caveolins-1 and -2 are normally co-expressed, and they form a hetero-oligomeric complex in many cell types. These caveolin hetero-oligomers are thought to represent the assembly units that drive caveolae formation in vivo. However, the functional significance of the interaction between caveolins-1 and -2 remains unknown. Here, we show that caveolin-1 co-expression is required for the transport of caveolin-2 from the Golgi complex to the plasma membrane. We identified a human erythroleukemic cell line, K562, that expresses caveolin-2 but fails to express detectable levels of caveolin-1. This allowed us to stringently assess the effects of recombinant caveolin-1 expression on the behavior of endogenous caveolin-2. We show that expression of caveolin-1 in K562 cells is sufficient to reconstitute the de novo formation of caveolae in these cells. In addition, recombinant expression of caveolin-1 allows caveolin-2 to form high molecular mass oligomers that are targeted to caveolae-enriched membrane fractions. In striking contrast, in the absence of caveolin-1 expression, caveolin-2 forms low molecular mass oligomers that are retained at the level of the Golgi complex. Interestingly, we also show that expression of caveolin-1 in K562 cells dramatically up-regulates the expression of endogenous caveolin-2. Northern blot analysis reveals that caveolin-2 mRNA levels remain constant under these conditions, suggesting that the expression of caveolin-1 stabilizes the caveolin-2 protein. Conversely, transient expression of caveolin-2 in CHO cells is sufficient to up-regulate endogenous caveolin-1 expression. Thus, the formation of a hetero-oligomeric complex between caveolins-1 and -2 stabilizes the caveolin-2 protein product and allows caveolin-2 to be transported from the Golgi complex to the plasma membrane.
Asunto(s)
Caveolinas , Membrana Celular/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Transporte Biológico , Northern Blotting , Caveolina 1 , Caveolina 2 , Humanos , Células K562 , ARN Mensajero/análisisRESUMEN
This study investigated the role of natural killer (NK) cells as effectors of an immune response against autologous cells modified by gene therapy. T lymphocytes were transduced with LXSN, a retroviral vector adopted for human gene therapy that carries the selectable marker gene neo, and the autologous NK response was evaluated. We found that (i) infection with LXSN makes cells susceptible to autologous NK cell-mediated lysis; (ii) expression of the neo gene is responsible for conferring susceptibility to lysis; (iii) lysis of neo-expressing cells is clonally distributed and mediated only by NK clones that exhibit human histocompatibility leukocyte antigen (HLA)-Bw4 specificity and bear KIR3DL1, a Bw4-specific NK inhibitory receptor; and (iv) the targets are cells from HLA-Bw4(+) individuals. Finally, neo peptides anchoring to the Bw4 allele HLA-B27 interfered with KIR3DL1-mediated recognition of HLA-B27, i.e., they triggered NK lysis. Moreover, neo gene mutations preventing translation of two of the four potentially nonprotective peptides reduced KIR3DL1(+) NK clone-mediated autologous lysis. Thus, individuals expressing Bw4 alleles possess an NK repertoire with the potential to eliminate autologous cells modified by gene therapy. By demonstrating that NK cells can selectively detect the expression of heterologous genes, these observations provide a general model of the NK cell-mediated control of viral infections.
Asunto(s)
Terapia Genética , Células Asesinas Naturales/inmunología , Secuencia de Aminoácidos , Células Clonales , Farmacorresistencia Microbiana/genética , Farmacorresistencia Microbiana/inmunología , Marcadores Genéticos/genética , Antígenos HLA-B/genética , Antígenos HLA-B/inmunología , Antígeno HLA-B27/genética , Antígeno HLA-B27/inmunología , Humanos , Kanamicina Quinasa/genética , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores KIR , Receptores KIR3DL1 , Retroviridae/genética , Linfocitos T/inmunologíaRESUMEN
Typical acute promyelocytic leukemia (APL) is associated with the t(15;17) translocation, expression of a PML/RARA fusion transcript, and responsiveness to all-trans retinoic acid (ATRA). Rare APL cases implicating the RARA but not the PML gene have been reported. Cases with t(11;17)(q23;q21) which fuses the PLZF and RARA genes do not respond to ATRA. In contrast, cases with t(11;17)(q13;q21) and t(5;17)(q35;q21) which fuse RARA with NuMA and NPM, respectively, were reported to be sensitive to ATRA. We described previously an APL case with an unbalanced t(5;17) implicating RARA but neither PML nor PLZF. Here, we show that in this case: (1) the NPM gene is not involved, as demonstrated by RT-PCR and Southern blot; (2) response to ATRA in vitro is atypical, as demonstrated by morphological and functional maturation assays; and (3) PML nuclear bodies are not disrupted, as evidenced by immunofluorescence staining.
Asunto(s)
Antineoplásicos/uso terapéutico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/genética , Tretinoina/uso terapéutico , Anciano , Antineoplásicos/farmacología , Núcleo Celular , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 5 , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Leucemia Promielocítica Aguda/patología , Receptores de Ácido Retinoico/genética , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Receptor alfa de Ácido Retinoico , Translocación Genética , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
The expression of the PML gene was investigated in purified early hematopoietic progenitor cells (HPCs) induced to unilineage erythroid or granulocytic differentiation. PML mRNA and protein, while barely detectable in quiescent HPCs, are consistently induced by growth factor stimulation through the erythroid or granulocytic lineage. Thereafter, PML is downmodulated in late granulocytic maturation, whereas it is sustainably expressed through the erythroid pathway. In functional studies, PML expression was inhibited by addition of antisense oligomers targeting PML mRNA (alpha-PML). Interestingly, early treatment (day 0 HPCs) with alpha-PML reduced the number of both erythroid and granulocytic colonies, whereas late treatment (day 5 culture) reduced erythroid, but not granulocytic, clonogenesis. These findings suggest that PML is required for early hematopoiesis and erythroid, but not granulocytic maturation. The pattern of PML expression in normal hematopoiesis mimics that of retinoblastoma pRb 105. Combined treatment of HPCs with alpha-PML and alpha-Rb oligomers inhibited both PML and Rb protein expression and completely blocked erythroid colony development. Furthermore, PML and pRb 105 were co-immunoprecipitated in cellular lysates derived from erythroid precursors indicating that this functional interaction may have a biochemical basis. These results suggest a key functional role of PML in early hematopoiesis and late erythropoiesis: the latter phenomenon may be related to the molecular and functional interaction of PML with pRb 105.
Asunto(s)
Hematopoyesis/genética , Proteínas de Neoplasias/fisiología , Proteínas Nucleares , Proteína de Retinoblastoma/fisiología , Factores de Transcripción/fisiología , Adulto , Diferenciación Celular , Regulación hacia Abajo , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Granulocitos/citología , Granulocitos/efectos de los fármacos , Humanos , Proteínas de Neoplasias/genética , Oligonucleótidos Antisentido/farmacología , Pruebas de Precipitina , Proteína de la Leucemia Promielocítica , ARN Mensajero/metabolismo , Proteína de Retinoblastoma/genética , Factores de Transcripción/genética , Proteínas Supresoras de TumorAsunto(s)
Regulación Leucémica de la Expresión Génica , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Leucemia/genética , Células Madre Neoplásicas/metabolismo , Apoptosis , Linaje de la Célula , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Proteína Ligando Fas , Humanos , Leucemia/patología , Glicoproteínas de Membrana/fisiología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/fisiología , Fenotipo , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento/fisiología , Receptores de Factores de Crecimiento Endotelial Vascular , Receptor fas/fisiologíaRESUMEN
The mechanisms responsible for persistence of T lymphocytes at the sites of allergic inflammation are not completely understood. Activated T cells, usually expressing Fas on their surface, undergo activation-induced apoptotic death, thus limiting the dangerous consequences of a persistent immune reaction. We have previously shown that pulmonary T lymphocytes from untreated asthmatic subjects do not express surface Fas receptors nor do they contain Fas mRNA, yet they display normal levels of Fas ligand. This is not an inherited defect and is confined to mucosal T cells. To gain insights into the mechanism responsible for these findings, we performed a set of experiments with both purified Dermatophagoides pteronyssinus allergen and recombinant human cytokines: interleukin 2 (IL-2), IL-4, IL-5, transforming growth factor beta1, interferon gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In vitro exposure of purified CD4(+) lymphocytes to allergen yielded only transient up-regulation of surface Fas but did not influence susceptibility to Fas-mediated cell death. T-helper type 2 cytokines (IL-4, IL-5, and GM-CSF) had a dose-dependent and specific inhibitory effect on Fas mRNA, suggesting a new fundamental biological role in the survival of inflammatory cells during allergen exposure.
Asunto(s)
Asma/inmunología , Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Receptores del Factor de Necrosis Tumoral/biosíntesis , Células Th2/inmunología , Receptor fas/biosíntesis , Adolescente , Adulto , Antígenos Dermatofagoides , Muerte Celular , Niño , Regulación hacia Abajo , Femenino , Glicoproteínas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Interferón gamma/inmunología , Interleucinas/inmunología , Activación de Linfocitos , Masculino , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
All-trans-retinoic acid (RA) treatment induces morphological remission in acute promyelocytic leukemia (APL) patients carrying the t(15;17) and expressing the PML/RARalpha product by inducing terminal differentiation of the leukemic clone. RA treatment induces downregulation of PML/RARalpha and reorganization of the PML-nuclear bodies. These events have been proposed to be essential for the induction of APL cell differentiation by RA. Here, we show that in the APL-derived NB4 cell line as well as in myeloid precursor U937 cells expressing the PML/RARalpha (U937/PR9) and in blasts from APL patients, the PML/RARalpha fusion protein is cleaved by a caspase 3-like activity induced by RA treatment. In fact, a caspase 3-like activity is detectable in PML/RARalpha expressing cells after RA treatment, and selective caspase inhibitor peptides are able to prevent the RA-induced degradation of the fusion protein in vivo and in vitro. Using recombinant caspases and PML/RARalpha deletion mutants we mapped a caspase 3 cleavage site (Asp 522) within the alpha-helix region of the PML component of the fusion protein. The extent of PML/RARalpha cleavage directly correlates with the ability of RA to restore the normal PML nuclear bodies (NBs) pattern. However, RA-induced differentiation is not prevented by the persistence of the fusion product and occurs in the absence of normally structured PML NBs. These results indicate that PML/RARalpha is directly involved in conferring RA sensitivity of APL cells and that the RA-induced reassembly of PML NBs is the consequence of the disappearance of PML/RARalpha.
Asunto(s)
Antineoplásicos/farmacología , Caspasas , Cisteína Endopeptidasas/fisiología , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Proteínas Nucleares , Proteínas de Fusión Oncogénica/metabolismo , Tretinoina/farmacología , Caspasa 3 , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Inducción Enzimática , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Promielocítica Aguda/patología , Complejos Multienzimáticos/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Proteína de la Leucemia Promielocítica , Complejo de la Endopetidasa Proteasomal , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
PML/RARalpha is the abnormal protein product of the Acute Promyelocytic Leukemia-specific 15;17 translocation. Both the PML and RARalpha components are required for the PML/RARalpha biological activities, namely its capacity to block differentiation and to increase survival of haematopoietic precursors. The physiological role of PML and its contribution to the function of the fusion protein are unknown. PML localizes to the cytoplasm and within specific nuclear bodies (NBs). In vitro, overexpression of PML correlates with suppression of cell transformation. The PML aminoterminal portion retained within the PML/RARalpha protein contains the RING finger, two newly defined cystein/histidine-rich motifs called B-boxes (B1 and B2) and a coiled-coil region. We report here that PML has a growth suppressive activity in all the cell lines tested, regardless of their transformed phenotype, and that the cellular basis for the PML growth suppression is induction of apoptotic cell death. Analysis of various nuclear and cytoplasmic PML isoforms showed that the PML growth suppressive activity correlates with its nuclear localization. Analysis of the localization and growth suppressive activity demonstrated that: (i) the Ring + B1-B2 and coiled-coil regions are both indispensable and sufficient to target PML to the NBs; (ii) individual deletions of the various PML domains have no effect on its growth suppressor activity; (iii) the Ring + B1-B2 region exerts a partial growth suppressor activity but its fusion with the coiled-coil region is sufficient to recapitulate the suppressive function of wild type PML. These results indicate that PML is involved in cell survival regulation and that the PML component of the fusion protein (Ring + B1-B2 and coiled-coil regions) retains intact biological activity, thereby suggesting that the effects of PML/RARalpha on survival derive from the activation of the incorporated PML sequence.
Asunto(s)
Apoptosis , Proteínas de Neoplasias/fisiología , Proteínas Nucleares , Factores de Transcripción/fisiología , Dedos de Zinc/fisiología , Células 3T3 , Animales , Sitios de Unión , División Celular , Línea Celular Transformada , Supervivencia Celular , Cisteína/genética , Cisteína/fisiología , Citoplasma/metabolismo , Células HeLa , Histidina/genética , Histidina/fisiología , Humanos , Isomerismo , Ratones , Mutagénesis , Proteínas de Neoplasias/genética , Proteína de la Leucemia Promielocítica , Factores de Transcripción/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Dedos de Zinc/genéticaRESUMEN
Acute promyelocytic leukemia (APL) is characterized by the t(15;17) cytogenetic abnormality leading to the expression of two fusion genes, PML/RARA and RARA/PML, and by its sensitivity to all-trans retinoic acid (ATRA) differentiating treatment. Rare APL cases lacking the t(15;17) have been described. We have previously reported two cases presenting with submicroscopic insertions of RARA or PML into chromosome 15 or 17, respectively. These insertions lead to the formation of potentially functional, nonreciprocal, PML/RARA or RARA/PML fusion genes, providing the unique opportunity to investigate in a human noncell-line model the respective role of PML/RARA or RARA/PML in retinoid signaling. Here, we report the in vitro response to ATRA of these two cases as well as of a third case presenting with submicroscopic insertion (15;17) and expressing exclusively PML/RARA, by morphological, functional, and immunological assays. The two cases expressing PML/RARA presented with an immunostaining pattern typical of APL and a positive response to ATRA, whereas the APL case expressing only a RARA/PML fusion transcript exhibited an immunostaining pattern typical of non-APL cells, and was resistant to ATRA. Our results confirm that sensitivity to ATRA requires expression of PML/RARA and strongly correlates with immunostaining, and demonstrate that expression of RARA/PML alone is sufficient for a cytological APL phenotype, but does not confer sensitivity to ATRA.
Asunto(s)
Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Translocación Genética , Tretinoina/farmacología , Adulto , Diferenciación Celular/efectos de los fármacos , Cromosomas Humanos Par 15/genética , Cromosomas Humanos Par 17/genética , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Conversión Génica/efectos de los fármacos , Conversión Génica/genética , Genes Relacionados con las Neoplasias , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/efectos de los fármacos , Proteínas de Fusión Oncogénica/metabolismo , Células Tumorales CultivadasRESUMEN
Acute promyelocytic leukaemia is characterized by translocations that involve the retinoic acid receptor alpha (RAR alpha) locus on chromosome 17 and the PML locus on 15 or the PLZF locus on 11. The resulting abnormal translocation products encode for PML/RAR alpha or PLZF/RAR alpha fusion proteins. There is increasing experimental evidence that the APL-specific fusion proteins have similar biologic activities on differentiation and survival and that both components of the fusion proteins (PML or PLZF and RAR alpha) are indispensable for these biological activities. The physiologic function of PML or PLZF or whether PML and PLZF contribute common structural or functional features to the corresponding fusion proteins is not known. We report here immunofluorescence studies on the cellular localization of PLZF and PLZF/RAR alpha and compare it with the localization of PML and PML/RAR alpha. PLZF localizes to nuclear domains of 0.3-0.5 microns, approximately 14 per cell in the KG1 myeloid cell line. These PLZF-bodies are morphologically similar to the domains reported for PML (PML-NBs). There is tight spatial relationship between about 30% of PLZ-NBs and PML-NBs: they partially overlap. However, PML and PLZF do not form soluble complexes in vivo. PLZF- and PML-NBs are functionally distinct. Adenovirus E4-ORF3 protein expression alters the structure of the PML-NBs and interferon increases the number of PML-NBs and neither has any effect on PLZF NBs. The localization of PLZF/RAR alpha is different to that of PLZF and RAR alpha. The nuclear distribution pattern of PLZF/RAR alpha is one of hundreds of small dots (microspeckles) less than 0.1 micron. Expression of PLZF/RAR alpha did not provoke disruption of the PML-NBs. Co-expression of PML/RAR alpha and PLZF/RAR alpha in U937 cells revealed apparent colocalization. Overall the results suggest that the PML- and PLZF-NBs are distinct functional nuclear domains, but that they may share common regulatory pathways and/or targeting sequences, as revealed by the common localization of their corresponding fusion proteins.
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Núcleo Celular/química , Proteínas de Unión al ADN/análisis , Proteínas de Neoplasias/análisis , Proteínas Nucleares/análisis , Factores de Transcripción/análisis , Técnica del Anticuerpo Fluorescente , Humanos , Factores de Transcripción de Tipo Kruppel , Proteína de la Leucemia Promielocítica , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Receptores de Ácido Retinoico/análisis , Proteínas Recombinantes de Fusión/análisis , Receptor alfa de Ácido Retinoico , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
We investigated the effect of the acute promyelocytic leukemia (APL) specific PML/RARalpha fusion protein on the sensitivity to TNF-alpha-mediated apoptosis. The U937 leukemia cell line was transduced with PML/RARalpha cDNA. PML/RARalpha expression caused a markedly reduced sensitivity to TNF-alpha, even if apoptosis was triggered by agonistic antibodies to TNF-alpha receptors I and II (TNF-alphaRI, II). PML/RARalpha induced a 10-20-fold decrease of the TNF-alpha-binding capacity via downmodulation of both TNF-alphaRI and TNF-alphaRII: this may mediate at least in part the reduced sensitivity to TNF-alpha. Furthermore, the fusion protein did not modify Fas expression (CD95) or sensitivity to Fas-mediated apoptosis. The pathophysiological significance of these findings is supported by two series of observations. (a) Fresh APL blasts exhibit no TNF-alpha binding and are resistant to TNF-alpha-mediated apoptosis. Conversely, normal myeloblasts-promyelocytes show marked TNF-alphaR expression and are moderately sensitive to TNF-alpha-mediated cytotoxicity. Similarly, blasts from other types of acute myeloid leukemia (AML M1, M2, and M4 FAB types) show an elevated TNF-alpha binding. (b) The NB4 APL cell line, which is PML/RARalpha+, shows low TNF-alphaR expression capacity and is resistant to TNF-alpha-triggered apoptosis; conversely a PML/RARalpha- NB4 subclone (NB4.306) exhibits detectable TNF-alpha-binding capacity and is sensitive to TNF-alpha-mediated cytotoxicity. These studies indicate that the PML/RARalpha fusion protein protects against TNF-alpha-induced apoptosis, at least in part via downmodulation of TNF-alphaRI/II: this phenomenon may play a significant role in APL, which is characterized by prolonged survival of leukemic blasts.
Asunto(s)
Apoptosis/fisiología , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Neoplasias/genética , Proteínas Nucleares , Receptores de Ácido Retinoico/genética , Proteínas Recombinantes de Fusión/fisiología , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/toxicidad , Antígenos CD/análisis , Antígenos CD/inmunología , División Celular/efectos de los fármacos , División Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Proteína de la Leucemia Promielocítica , Unión Proteica/efectos de los fármacos , ARN Mensajero/análisis , Receptores del Factor de Necrosis Tumoral/agonistas , Receptores del Factor de Necrosis Tumoral/inmunología , Receptor alfa de Ácido Retinoico , Transfección , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Receptor fas/metabolismoRESUMEN
BACKGROUND: Bronchial fistula is one of the most serious complications of pulmonary resection. METHODS: We present an endoscopic treatment that consists of multiple submucosal injections of polidocanol-hydroxypoliethoxidodecane (Aethoxysklerol Kreussler) on the margins of the fistula using an endoscopic needle inserted through a flexible bronchoscope. RESULTS: From 1984 to 1995, 35 consecutive nonselected patients with a postresectional bronchopleural fistula were treated. All 23 partial postpneumonectomy or postlobectomy bronchopleural fistulas, ranging from 2 to 10 mm in diameter, healed completely. This did not occur in the 12 total bronchial dehiscences. No complications occurred due to the injection of the drug. CONCLUSIONS: In our opinion this treatment can be considered a valid therapeutic approach, as it is simple, safe, scarcely traumatic, and inexpensive, particularly considering that, in patients in stable condition, it can be performed as an outpatient treatment.