RESUMEN
Patients with myelodysplastic syndrome (MDS) have ineffective in vivo and in vitro erythropoiesis, characterized by an impaired response to erythropoietin (Epo). We examined proliferation and maturation of MDS marrow cells in response to Epo in more detail. Epo-dependent DNA synthesis as well as induction of GATA-1 binding activity in marrow cells from 15 MDS cases were severely reduced as compared with normal bone marrow (NBM). Additionally, the appearance of morphologically identifiable erythroid cells was decreased in MDS cell cultures. These data indicate that both the Epo-dependent proliferation as well as the differentiation induction by Epo is suppressed. To study more upstream events of the Epo signal transduction route we investigated activation of the signal transducer and activator of transcription (STAT) 5. In all 15 MDS samples tested, STAT5 activation was absent or greatly suppressed in response to Epo. In contrast, interleukin-3 induced a normal STAT5 response in MDS cells. Further, in MDS the subset of CD71+ BM cells that is phenotypically similar to Epo-responsive cells in normal marrow, was present. We conclude that the Epo response in MDS is disturbed at an early point in the Epo-receptor (EpoR) signal transduction pathway.
Asunto(s)
Médula Ósea/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Eritropoyetina/farmacología , Proteínas de la Leche , Síndromes Mielodisplásicos , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Médula Ósea/metabolismo , Médula Ósea/patología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Factores de Unión al ADN Específico de las Células Eritroides , Femenino , Factor de Transcripción GATA1 , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Factor de Transcripción STAT5RESUMEN
Interleukin-7 (IL-7) stimulates the proliferation of normal and leukemic B and T cell precursors and T lymphocytes. Activation of the JAK/STAT pathway has been implicated in IL-7R signaling. We investigated which STAT complexes are formed upon stimulation of B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells with IL-7. Gel retardation assays with STAT-binding oligonucleotides showed that IL-7 induces the formation of two major STAT complexes in BCP-ALL cells. Supershifts with anti-STAT antibodies identified these as STAT1 and STAT5 complexes. This pattern of STAT activation was seen in all BCP-ALL cases that respond to IL-7 in proliferation assays. IL-7 also induced STAT/DNA binding in BCP-ALL cases that failed to proliferate in response to IL-7, suggesting that the ability of IL-7R to activate the JAK/STAT pathway per se is not sufficient for proliferation induction. To determine the contribution of the cytoplasmic domain of the IL-7 receptor alpha chain (IL-7R alpha) to activation of STAT proteins, transfectants of the murine pro-B cell line BAF3 were made that express chimeric receptors consisting of the extracellular domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) and the transmembrane and intracellular domains of human IL-7R alpha. Activation of the chimeric G-CSF-R/IL-7R alpha with G-CSF resulted in a full proliferative response and induced the phosphorylation of JAK1 but not JAK2. Major STAT complexes activated by G-CSF-R/IL-7R alpha contained STAT1 or STAT5, while some formation of STAT3-containing complexes was also seen. These findings establish that STAT1 and STAT5, and possibly STAT3, are activated upon stimulation of precursor B cells with IL-7. The data further indicate that the IL-7R alpha chains are directly involved in the activation of JAKs and STATs and have a major role in proliferative signaling in precursor B cells.
Asunto(s)
Antígenos CD/fisiología , Linfocitos B/fisiología , Proteínas de Unión al ADN/metabolismo , Interleucina-7/farmacología , Proteínas de la Leche , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Proteínas Proto-Oncogénicas , Receptores de Interleucina/fisiología , Transducción de Señal , Transactivadores/metabolismo , Animales , Antígenos CD/biosíntesis , Antígenos CD/química , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Secuencia de Bases , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/fisiopatología , Línea Celular , Cartilla de ADN , Activación Enzimática , Humanos , Janus Quinasa 1 , Janus Quinasa 2 , Cinética , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Proteínas Tirosina Quinasas/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/biosíntesis , Receptores de Interleucina/biosíntesis , Receptores de Interleucina/química , Receptores de Interleucina-7 , Proteínas Recombinantes de Fusión/biosíntesis , Factor de Transcripción STAT1 , Factor de Transcripción STAT5 , Transducción de Señal/efectos de los fármacos , Timidina/metabolismo , Células Tumorales CultivadasRESUMEN
A 44-year-old man suffering from cytogenetically and molecularly proven Philadelphia translocation-positive chronic myelogenous leukemia in chronic phase was treated with busulfan for 18 months and studied during a follow-up period of 13 years. Hematologically and cytogenetically, he attained a continuing complete remission, although at one point (9.5 years) at least, after attaining complete remission molecular analysis indicated the presence of minimal residual disease.
Asunto(s)
Antineoplásicos Alquilantes/uso terapéutico , Busulfano/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Adulto , Sangre/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Humanos , Cariotipificación , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Masculino , Inducción de RemisiónRESUMEN
Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) involves the activation of the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway. G-CSF induces tyrosine phosphorylation of Jak1, Jak2, STAT1, and STAT3. The membrane-proximal region of G-CSF-R is sufficient for activation of Jaks. It is still unclear how STAT proteins are activated by G-CSF-R. We investigated the possible involvement of the C-terminal region of G-CSF-R in the recruitment of STAT proteins using BAF3 cell transfectants expressing wild type (WT) G-CSF-R, C-terminal deletion mutants and tyrosine-to-phenylalanine substitution mutants. Electrophoretic mobility shift assays with STAT-binding oligonucleotides (m67) showed that activation of WT G-CSF-R induces three distinct STAT complexes, namely STAT3 homodimers, STAT1-STAT3 heterodimers, and STAT1 homodimers. However, STAT1 homodimers and STAT1-STAT3 heterodimers were predominantly formed after activation of a C-terminal deletion mutant d685, which lacks all four conserved cytoplasmic tyrosine residues, located at positions 704, 729, 744, and 764. Antiphosphotyrosine immunoblots of STAT3 immunoprecipitates showed that activation of WT G-CSF-R induced phosphorylation of STAT3. In contrast, no phosphorylation of STAT3 was observed after activation of deletion mutant d685. These findings establish that the C-terminal region of G-CSF-R plays a major role in the activation of STAT3. By using tyrosine-to-phenylalanine substitution mutants of G-CSF-R, we further show that tyrosine 704, present in a YXXQ consensus sequence shown to be essential for STAT3 binding to gp130, is not exclusively involved in the activation of STAT3 by G-CSF-R.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Receptores de Factor Estimulante de Colonias de Granulocito/fisiología , Transactivadores/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Secuencia de Consenso , Citoplasma , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Fosfotirosina/metabolismo , Unión Proteica , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transducción de SeñalRESUMEN
Recently we reported cytogenetic, clinical, and immunologic data of 135 childhood ALL patients, who were diagnosed and treated in The Sophia Children's Hospital between January 1, 1980 and November 1, 1990. An increased risk for a first relapse in the central nervous system (CNS) was detected in a subgroup of childhood ALL patients with common ALL or pre-B ALL phenotype and chromosomal aberrations of the short arm of chromosome 12. In this paper we report clinical, cytogenetic, immunologic, morphologic and cytochemical data on these eight childhood ALL patients with aberrations of the short arm of chromosome 12 and of an additional four cases that were diagnosed and treated between November 1, 1990 and February 1, 1992. We found that three out of six common ALL, two out of three pre-B ALL and one out of three T-ALL patients with 12p chromosomal rearrangements developed a first relapse in the CNS. On the contrary, the frequency of CNS relapse in our childhood ALL patients without 12p aberrations was 10%. Furthermore, morphologic and cytochemical analysis of the bone marrow smears of these 12 patients with aberrations of the short arm of chromosome 12 revealed that the nine cases with pre-B or common ALL phenotype had typical morphologic characteristics that are unusual for newly diagnosed childhood ALL. Typical for this subtype is the presence of large polymorphic blast cells without nucleoli. The nuclei are irregularly shaped showing folds and clefts and a stripy pattern. The nucleus and cytoplasm are often abundantly vacuolated. The cytoplasm has a foamy light-blue appearance.
Asunto(s)
Médula Ósea/patología , Neoplasias del Sistema Nervioso Central/secundario , Aberraciones Cromosómicas , Cromosomas Humanos Par 12 , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Núcleo Celular/patología , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/inmunología , Niño , Preescolar , Citoplasma/patología , Femenino , Humanos , Inmunofenotipificación , Lactante , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , PronósticoRESUMEN
We report two patients with a myeloproliferative disorder (Philadelphia chromosome-negative chronic myeloid leukemia) and t(5;12)(q31;p12). Until now, only three cases of a translocation (5;12)(q31;p12) have been reported. All investigators had problems classifying their patient's disease into one of the well-defined entities of either MPD or myelodysplastic disorders. We postulate that this translocation may represent a subgroup of patients with features of both chronic myeloid leukemia and chronic myelomonocytic leukemia (CMMoL).
Asunto(s)
Cromosomas Humanos Par 12 , Cromosomas Humanos Par 5 , Leucemia Mieloide Crónica Atípica BCR-ABL Negativa/genética , Leucemia Mielomonocítica Crónica/genética , Translocación Genética , Adulto , Southern Blotting , Humanos , Cariotipificación , Masculino , Persona de Mediana EdadRESUMEN
The translocation (6;9)(p23;q34) is mainly found in specific subtypes of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The diagnosis of this translocation is not easy since the cytogenetic change is quite subtle. The two genes involved in this translocation were recently isolated and diagnosis at the DNA-level became an additional option. Both the dek gene on chromosome 6 and the can gene on chromosome 9 contain one specific intron where breakpoints of t(6;9) patients were found to cluster. The translocation results in a consistent chimeric dek-can mRNA which is generated from the 6p- derivative. Five centers participated in a study to estimate the incidence of t(6;9) in leukemic patients using conventional Southern blot analysis. Patients (n = 320) with either acute undifferentiated leukemia (AUL), AML, MDS or acute lymphoblastic leukemia (ALL) were screened for rearrangement of the genes involved in this translocation. Four of these 320 patients showed rearrangement of the can gene on chromosome 9, of which one also had a rearranged dek gene on chromosome 6. A further 20 patients were studied with karyotypic aberrations in which either the short arm of chromosome 6 or the long arm of chromosome 9 were specifically involved. Both conventional Southern blot analysis and contour-clamped homogeneous electric field (CHEF) analysis failed to show dek-can rearrangement in any of these patients. The results of our study indicate that the incidence of the t(6;9) is a low as reported based on cytogenetic data and that rearrangement of the dek and can genes is mainly restricted to this specific translocation.
Asunto(s)
Cromosomas Humanos Par 6 , Cromosomas Humanos Par 9 , Reordenamiento Génico , Leucemia/genética , Translocación Genética , Enfermedad Aguda , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Southern Blotting , Niño , Preescolar , Fragilidad Cromosómica , Mapeo Cromosómico , Electroforesis/métodos , Femenino , Humanos , Cariotipificación , Masculino , Persona de Mediana Edad , Familia de Multigenes , Síndromes Mielodisplásicos/genéticaRESUMEN
Acute lymphoblastic leukemia (ALL) is the most frequent childhood cancer. The disease is heterogeneous. The leukemic cells in childhood ALL patients show various karyotypic abnormalities, express diverse phenotypes, and respond variably to treatment. Identification of prognostic factors will make it possible to predict treatment outcome and to identify the patients that require different therapeutic approaches. The important prognostic implications of chromosome number and of the presence of several well defined structural chromosomal aberrations have been established by several groups. We analyzed 145 children with ALL at diagnosis for cytogenetic features. In 135 cases cytogenetic analysis was successful. Of these, 101 showed abnormal karyotype (70%). Structural chromosomal rearrangements were detected in 71 out of 135 cases investigated (53%), i.e. 71% of the cytogenetically abnormal cases. These cytogenetic findings were correlated with clinical outcome. In our series the ploidy of the karyotype appeared to be of prognostic importance. Our findings concerning karyotype, phenotype, and treatment outcome of this subgroup were in most cases in agreement with earlier reports from other investigators. We observed an increased risk for central nervous system relapse in childhood ALL patients with abnormalities involving the short arm of chromosome 12 in combination with pre-B or common ALL phenotype.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Niño , Preescolar , Aberraciones Cromosómicas , Femenino , Estudios de Seguimiento , Humanos , Inmunofenotipificación , Lactante , Cariotipificación , Masculino , Ploidias , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Pronóstico , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Using the polymerase chain reaction and Southern blot analysis the expression was detected of a bcr-abl mRNA lacking abl exon a2. This was due to a corresponding unusual localization of the breakpoint in the c-abl gene and was seen in a patient with Philadelphia (Ph) chromosome positive chronic myelogeneous leukemia in chronic phase. This type of mRNA has been described only once before in two Ph-positive acute lymphoblastic leukemia patients, by Soekarman et al. (1). The abl exon a2 sequences, which are missing in the three reported patients, code for a part of the SH3 region of the abl protein, which is supposed to be involved in negative regulation of the kinase domain. The clinical significance of this finding is discussed.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 9 , Proteínas de Fusión bcr-abl/genética , Genes abl , Leucemia Mieloide de Fase Crónica/genética , ARN Mensajero/análisis , Southern Blotting , Exones , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mieloide de Fase Crónica/metabolismo , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
Between 1985 and 1989, many cases of Philadelphia (Ph) chromosome negative chronic myelogenous leukemia (CML) were reported. For this review, the following selection criteria were used: the original articles on Ph-negative cases should provide clinical, hematologic, cytogenetic as well as molecular data. In addition, eight unpublished cases of Ph-negative CML are included that were studied in our institute during the last two years. Our purpose was to correlate presence or absence of the Ph rearrangement with the clinical features in an attempt to test whether the entity "Ph-negative CML" really exists and to identify the pathologic characteristics, frequency of occurrence, prognosis for survival, and underlying molecular mechanisms. Data on Ph-negative CML patients were compared with data on Ph-positive CML, atypical CML (aCML), and chronic myelomonocytic leukemia (CMMoL), reported in the same papers as the Ph negative patients. Essential for comparison of data from the different investigators appeared to be a clear description of criteria they used to establish the diagnosis CML, or alternatively a complete presentation of data for all patients reported in the articles. In most cases, Ph-negative CML was distinguishable from CMMoL and aCML, using simple criteria, e.g., differential count of peripheral blood and absence of dysplasia in the bone marrow. Cytogenetic analysis showed normal karyotype in most cases of Ph-negative CML. Interestingly, in cases with abnormal karyotype, chromosome 9 band q34 was relatively frequently involved in translocations with other chromosomes than chromosome 22, suggesting a variant Ph translocation not visible by cytogenetic techniques. This assumption was confirmed by molecular analysis, demonstrating bcr-abl rearrangement in 9 out of 10 of the latter cases. Results of cytogenetic and molecular investigations in 136 cases of Ph-negative CML reviewed in this article clearly indicated that molecular techniques are valuable tools for identification of bcr-abl rearrangements, indicative for the Ph translocation. The different mechanisms responsible for bcr-abl rearrangement in Ph-negative CML patients are discussed. The question remains whether all Ph-negative CML patients will have bcr-abl rearrangements, or whether alternative mechanisms will be identified that are responsible for this disease.
Asunto(s)
Leucemia Mieloide Crónica Atípica BCR-ABL Negativa/genética , Cromosoma Filadelfia , Cromosomas Humanos Par 22 , Cromosomas Humanos Par 9 , Femenino , Proteínas de Fusión bcr-abl/genética , Genes abl , Humanos , Cariotipificación , Leucemia Mieloide Crónica Atípica BCR-ABL Negativa/patología , Masculino , Translocación GenéticaRESUMEN
As a consequence of the Ph translocation, a bcr-abl fusion gene is formed that encodes chimeric bcr-abl proteins. The latter are probably causally involved in leukemogenesis, although the mechanism is not yet known. Meanwhile, the heterogeneous aspects of the Ph translocation have been explored in an effort to disclose their eventual clinical significance. Innovative and useful methods have been devised for the detection of the Ph translocation products e.g. the PCR technique or the production of specific antibodies that undoubtedly will prove to be of great help to patients and clinicians in future.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocación Genética/genética , Mapeo Cromosómico , Humanos , Pruebas Inmunológicas , Leucemia Mieloide Crónica Atípica BCR-ABL Negativa/genética , Reacción en Cadena de la Polimerasa , Proto-Oncogenes/fisiologíaRESUMEN
The diagnostic and prognostic value of specific cytogenetic abnormalities has been established for most recurrent translocations. For less frequent changes, we still need to collect more cases for determination of their clinical significance. Optimal treatment of leukemia with modern therapeutic strategies requires knowledge of the prognostic factors, and leukemic karyotype should be one of the variable features systematically evaluated in all trials. The molecular analysis of the specific translocation will considerably increase our understanding of the mechanism of leukemogenesis and provide us with new tools for diagnosis. Systematic collection and conservation of acute leukemic cells, cytogenetically and immunologically characterized, would greatly facilitate and accelerate these fundamental studies.
Asunto(s)
Marcadores Genéticos , Técnicas Genéticas , Leucemia/genética , Enfermedad Aguda , Adulto , Niño , Aberraciones Cromosómicas , Reordenamiento Génico , Humanos , Leucemia/diagnóstico , Leucemia/patología , PronósticoRESUMEN
We report here the case of a patient with refractory anemia with excess of blasts (RAEB) which evolved into RAEB in transformation. The standard Philadelphia (Ph) chromosome was found by cytogenetic study at diagnosis and during evolution. Southern blot analysis showed breakpoint cluster region (bcr) rearrangement as observed in chronic myelogenous leukemia (CML).
Asunto(s)
Anemia Refractaria con Exceso de Blastos/genética , Cromosoma Filadelfia , Anciano , Anciano de 80 o más Años , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , MasculinoRESUMEN
We studied the clinical, hematologic, cytogenetic and molecular biologic features in four patients with Philadelphia (Ph) negative chronic myeloid leukemia (CML). In all four cases the clinical and hematologic characteristics were indistinguishable from Ph positive CML. Cytogenetic analysis showed a normal karyotype in two patients and chromosomal translocations apparently not affecting chromosome 22 in the other two cases. Southern blot analysis using probes of the bcr region, demonstrated a bcr break-point in all four patients. In situ hybridization with bcr, c-abl, and c-sis probes showed unusual hybridization sites for 5'-bcr and c-abl indicating complex chromosomal rearrangements affecting three different chromosomes in the four patients investigated. Using polymerase chain reaction (PCR) followed by hybridization to oligonucleotide probes specific for the bcr-abl fusion region, the expression of a chimeric bcr-abl mRNA was detected. In these patients we demonstrated that (a) CML with a breakpoint in the bcr region without cytogenetically detectable Ph chromosome is characterized by the same genomic recombination of 5'-bcr and c-abl as CML with standard Ph translocation and (b) unusual localization of 5'-bcr and c-abl sequences caused by complex Ph translocation does not interfere with transcription of the bcr-abl fusion gene.