RESUMO
It remains unclear in adult acute myeloid leukaemia (AML) whether leukaemic expression of CD33, the target antigen for gemtuzumab ozogamicin (GO), adds prognostic information on GO effectiveness at different doses. CD33 expression quantified in 1583 patients recruited to UK-NCRI-AML17 (younger adults) and UK-NCRI-AML16 (older adults) trials was correlated with clinical outcomes and benefit from GO including a dose randomisation. CD33 expression associated with genetic subgroups, including lower levels in both adverse karyotype and core-binding factor (CBF)-AML, but was not independently prognostic. When comparing GO versus no GO (n=393, CBF-AMLs excluded) by stratified subgroup-adjusted analysis, patients with lowest quartile (Q1) %CD33-positivity had no benefit from GO (relapse risk, HR 2.41 (1.27-4.56), P=0.009 for trend; overall survival, HR 1.52 (0.92-2.52)). However, from the dose randomisation (NCRI-AML17, n=464, CBF-AMLs included), 6 mg/m2 GO only had a relapse benefit without increased early mortality in CD33-low (Q1) patients (relapse risk HR 0.64 (0.36-1.12) versus 1.70 (0.99-2.92) for CD33-high, P=0.007 for trend). Thus CD33 expression is a predictive factor for GO effect in adult AML; although GO does not appear to benefit the non-CBF AML patients with lowest CD33 expression a higher GO dose may be more effective for CD33-low but not CD33-high younger adults.
Assuntos
Aminoglicosídeos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/análise , Adolescente , Adulto , Fatores Etários , Aminoglicosídeos/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Biomarcadores/análise , Relação Dose-Resposta a Droga , Feminino , Gemtuzumab , Humanos , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Recidiva , Taxa de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
Although the prognostic impact of mutations of FLT3 and NPM1 have been extensively studied in younger patients with acute myeloid leukaemia, less is known in older patients whether treated intensively or non-intensively, or in the context of existing prognostic scores. In 1312 patients 16 and 21%, respectively had an FLT3 and NPM1 mutation. An FLT3 mutation did not affect remission rate in intensively or non-intensively treated patients but was associated with an inferior survival. All patients with an NPM1c mutation had a significantly higher remission rate irrespective of treatment approach but survival was not improved, overall, or in any genotype except as in younger patients, in the FLT3 WT NPM1c mutant subgroup. When incorporated into an established multi-parameter prognostic risk score, the molecular information provided additional prognostic definition in 11% of patients.
Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mutação , Proteínas Nucleares/genética , Tirosina Quinase 3 Semelhante a fms/genética , Idoso , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Prognóstico , Recidiva , Indução de Remissão , Risco , Resultado do Tratamento , Reino UnidoRESUMO
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Regulação Leucêmica da Expressão Gênica/genética , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/fisiologia , Transcrição Gênica/genética , Antígenos CD/biossíntese , Antígenos CD/genética , Linhagem Celular Tumoral/metabolismo , Linhagem da Célula , Células Cultivadas/metabolismo , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 21/ultraestrutura , Cromossomos Humanos Par 8/genética , Cromossomos Humanos Par 8/ultraestrutura , Desmoplaquinas/genética , Desmoplaquinas/fisiologia , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/patologia , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Leucemia Mieloide Aguda/patologia , Proteínas de Neoplasias/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteína 1 Parceira de Translocação de RUNX1 , Receptores de Interleucina-17/biossíntese , Receptores de Interleucina-17/genética , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Transcrição SOXC , Transativadores/biossíntese , Transativadores/genética , Translocação Genética , gama Catenina/genética , gama Catenina/fisiologiaRESUMO
The Philadelphia (Ph) chromosome, a characteristic cytogenetic marker of chronic myeloid leukaemia (CML), is caused by a reciprocal translocation juxtaposing the 3' region of the ABL gene onto the 5' region of the BCR gene. Due to conservation of the reading frame, but depending on the site of the breakpoint in the BCR gene, two alternatively spliced variants of the p210BCR-ABL mRNA (known as b2-a2 and b3-a2) are produced. To investigate whether there are any biological differences between these splice variants we have transfected the b3-a2 or b2-a2 cDNA into a murine myeloid cell line, 32D. We have also included the previously prepared 32Dp210 cell line (which expresses the b3-a2 transcript) in all of our comparisons. RT-PCR analysis indicated that transcription levels were comparable between the variants. Morphological examination of the cells expressing either of the BCR-ABL transcripts indicated that these cells were more mature with increased cytoplasm:nuclear ratios compared to the 32D parental and 32Dneo vector control cells. However, the 32Dp210 cells had a very different appearance from the other panel members and flow karyotyping indicated a clonal evolution and cytogenetic instability in these cells alone. At 10(6) and 10(7) cell doses all 32D cells expressing BCR-ABL caused ill health and tissue infiltration in SCID mice with such rapidity that statistical analysis was not informative. However, at the 10(5) and 10(4) dosage levels there were similar survival rates between mice injected with 32Db2-a2 or 32Db3-a2 while mice injected with 32Dp210 had a significantly shorter survival time. The study of this 32D cell line panel indicated that there were no overt differences in the biological properties conferred by the b3-a2 or b2-a2 transcripts to the 32D cells although these transcripts were able to confer in vitro and in vivo biological effects. This panel of BCR-ABL expressing 32D cells provides a useful model for CML disease progression studies.
Assuntos
Processamento Alternativo/genética , Proteínas de Fusão bcr-abl/administração & dosagem , Proteínas de Fusão bcr-abl/genética , Animais , Divisão Celular , Linhagem Celular/transplante , Movimento Celular , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Injeções Intravenosas , Cariotipagem , Camundongos , Camundongos SCID , Isoformas de Proteínas/administração & dosagem , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Retinoic acid has the ability to induce differentiation in some myeloid leukaemia cell lines and has been used to induce remission in acute promyelocytic leukaemia patients. We have analysed changes in gene expression, by differential display, in HL60 cells exposed to all-trans retinoic acid (ATRA) for only 1 h. Only about 0.4% of the genes examined by this technique showed changes in expression level, and all four of the gene fragments identified were downregulated during the short 1 h exposure. Two of the fragments were novel, a third was MYC and the fourth was the FUS proto-oncogene. Northern analysis showed that FUS was downregulated within 1 h only during induced neutrophil differentiation but not at all during induced monocyte differentiation. Unlike the sensitive cell lines, ATRA-resistant cell lines did not show a downregulation of FUS over a 24 h period of exposure to ATRA. Using a semiquantitative PCR analysis, no difference in FUS levels was observed between ATRA-sensitive and -resistant cell lines. A similar analysis was carried out on primary acute myeloid leukaemia (AML), peripheral stem cell harvests (PBSC) and cord blood samples. The PBSC and cord blood samples had FUS levels that were similar or generally less than the cell lines. However, much higher levels were seen in 63% of the AML samples examined. The data presented are consistent with previous reports for a role for FUS in the promotion and maintenance of cellular proliferation.
Assuntos
Leucemia Mieloide/metabolismo , Ribonucleoproteínas/metabolismo , Doença Aguda , Diferenciação Celular , Células HL-60 , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Proto-Oncogene Mas , Proteína FUS de Ligação a RNA , Tretinoína/farmacologiaRESUMO
The myelomonocytic cell line HL60 can be induced by a variety of chemical agents to differentiation to either neutrophils or monocytes. Examination of gene expression, by differential display, in cells induced to monocytes with 1alpha,25-dihydroxyvitamin D(3) or neutrophils with all-trans retinoic acid (ATRA) identified a number of clones with altered patterns of expression over the period of differentiation. One of these clones was the mitochondrial gene NADH dehydrogenase subunit 4 (ND4) which showed a differential pattern of expression between the neutrophil and monocyte lineages. The potential of mitochondrial inhibitors to induce differentiation was investigated by treating the HL60 cells with either the NADH dehydrogenase inhibitor, Rotenone, the complex III inhibitor, Antimycin A, or the highly specific mitochondrial ATP-synthase inhibitor, Oligomycin. Although functional assays of differentiation did not produce any positive results, all the inhibitors resulted in a dramatic increase in CD14 expression at day 1, with CD38 markers not observed until day 3. The increased expression of CD14 was accompanied by a decrease in viability and all CD14 positive cells were also positive for Annexin V, a marker of apoptosis. These results suggest that inhibition of the components of the mitochondrial pathways may lead to the marking of some cells, via CD14, for cell death, whilst allowing commitment to differentiation to occur in the surviving population.
Assuntos
Antígenos CD , Apoptose , Células HL-60/efeitos dos fármacos , Leucócitos/citologia , Receptores de Lipopolissacarídeos/biossíntese , Mitocôndrias/efeitos dos fármacos , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Anexina A5/biossíntese , Antígenos de Diferenciação/biossíntese , Antimicina A/farmacologia , Calcitriol/farmacologia , Diferenciação Celular , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Glicoproteínas de Membrana , Monócitos/citologia , NAD+ Nucleosidase/biossíntese , NADH Desidrogenase/antagonistas & inibidores , Neutrófilos/citologia , Oligomicinas/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Rotenona/farmacologia , Tretinoína/farmacologia , Desacopladores/farmacologiaRESUMO
Neutrophil and monocyte differentiation can be induced in HL60 leukaemia cells by all-trans-retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3), respectively, whose differentiating effects can be enhanced by exposure to 'anti-inflammatory agents' and steroids. We have provided evidence that this potentiation is via inhibition of the activity of an enzyme of the aldoketoreductase (AKR) family, but had failed to identify expression of known AKRs in HL60 cells. In this study, we have identified a previously unclassified aldoketoreductase family member (termed HAKR e) that is expressed in HL60 cells. HAKR e is dramatically and transiently up-regulated in HL60 cells within 24 h of exposure to ATRA, further supporting the proposition that a member(s) of this family of enzymes play(s) a role in controlling cell growth and/or differentiation.
Assuntos
Regulação Enzimológica da Expressão Gênica , Monócitos/enzimologia , Neutrófilos/enzimologia , Tretinoína/farmacologia , Northern Blotting , Western Blotting , Calcitriol/farmacologia , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Biblioteca Gênica , Células HL-60 , Humanos , Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Indometacina/farmacologia , Monócitos/citologia , Neutrófilos/citologia , Fagocitose , Mapeamento por Restrição , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A recent clinical therapeutic initiative has been the use of chemical agents which induce the leukaemic cells to overcome their block in differentiation. In order to understand this block the cascade of molecular events needs to be characterized. Haemopoietic differentiation is ultimately controlled at the level of gene transcription which is mediated by an array of transcription factors. Many transcription factors contain similar structural protein sequences, and we have used an RT-PCR-based approach to isolate sequences, from transcription factor gene families which share similar domains. Degenerate primers corresponding to the TFIIIA zinc-finger consensus amino acid sequences and to the POU-homeodomain and POU-specific domain were used to amplify genes on the basis that they contained similarities in structural motifs shared within these families of transcription factors. A serum-independent HL60 cell line was induced towards the neutrophil lineage by treatment with all-trans retinoic acid (ATRA) for 24 h. CD38+ cells committed towards this lineage were enriched and a population of these cells treated with dihydroxyvitamin D3 to induce neutrophil maturation. RNA extracted from uninduced, ATRA-induced CD38+ cells, and vitamin D3 treated maturing cell cultures were amplified using the degenerate primers. PCR fragments were cloned, sequenced, clustered into homologous groups, and the group sequences searched on the GenBank database. The Oct 1 transcription factor, and a very close homologue, KIAA0144, was identified using the POU family primers. The zinc-finger primers identified three zinc-finger genes. The pattern of gene expression was suggested from the number of clones in each group at neutrophil commitment and maturation. The differential expression of the genes in the zinc finger and POU families will lead to a better understanding of the cascade of gene expression which occurs following ATRA-induced differentiation.
Assuntos
Neutrófilos/patologia , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Clonais , Células HL-60 , Humanos , Neutrófilos/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Transcrição , Dedos de ZincoRESUMO
Preincubation of AtT-20 mouse pituitary tumour cells with the phorbol ester PMA resulted in a concentration-dependent inhibition of CNP-stimulated cyclic GMP production. The phorbol ester analogue 4 alpha phorbol had no inhibitory effect and 24 h preincubations with PMA resulted in a characteristic down-regulation of the response indicating that the inhibitory actions were mediated via the activation of protein kinase C. Forskolin in the presence of the phosphodiesterase inhibitor IBMX stimulated intracellular cyclic AMP concentrations by up to eight fold, but did not alter basal nor CNP-stimulated cyclic GMP production. These results indicate that CNP-stimulated guanylate cyclase activity associated with the GC-B natriuretic peptide receptor expressed in AtT-20 cells is inhibited by protein kinase C.
Assuntos
GMP Cíclico/metabolismo , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Fator Natriurético Atrial/farmacologia , Linhagem Celular , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Cinética , Camundongos , Peptídeo Natriurético Tipo C , Neoplasias Hipofisárias , Proteínas/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Receptors for the natriuretic peptide family have been characterized in the adrenocorticotrophic hormone (ACTH)-secreting AtT-20 pituitary tumour cell line. Northern blot analysis detected mRNA transcripts for the guanylate cyclase-linked GC-B receptor subtype. There was no evidence for the expression of either guanylate cyclase-linked GC-A receptor or atrial natriuretic peptide (ANP)-C (clearance) receptor mRNAs. Cyclic GMP production in AtT-20 cells was stimulated up to 200-fold by C-type natriuretic peptide (CNP), which was 10- and 20 times as effective as equivalent concentrations of brain natriuretic peptide and ANP respectively. Cyclic GMP dose-response curves to CNP failed to show any signs of saturation even at concentrations up to 30 microM, indicating a relatively low affinity of CNP for the GC-B receptor. Although CNP induced large stimulations in cyclic GMP production, specific binding of [125I-Tyr0]CNP could not be demonstrated in AtT-20 cells. The absence of specific binding with this radiolabelled analogue is possibly due to a reduced affinity for the GC-B receptor, as CNP analogues with N-terminal modifications such as [Tyr0]CNP and [127I-Tyr0]CNP exhibited reduced abilities to stimulate cyclic GMP production in these cells. Despite elevating cyclic GMP levels, CNP had no effect on basal or corticotrophin-releasing factor-stimulating ACTH release from the cells. These results show that the guanylate cyclase-coupled GC-B receptor is the only natriuretic peptide receptor subtype expressed in AtT-20 cells. Although CNP can markedly stimulate cyclic GMP production in these cells, there is incomplete expression of the normal natriuretic peptide-induced inhibitory pathway of ACTH secretion at some point distal to the production of cyclic GMP.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Fator Natriurético Atrial/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Neoplasias Hipofisárias/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Animais , Fator Natriurético Atrial/metabolismo , Linhagem Celular , Hormônio Liberador da Corticotropina/farmacologia , GMP Cíclico/metabolismo , Cinética , Peptídeo Natriurético Tipo C , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Receptores do Fator Natriurético Atrial/biossíntese , Células Tumorais CultivadasRESUMO
Eleven green individuals were isolated when 95000 M2 plants of barley (Hordeum vulgare L.), mutagenised with azide in the M1, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F2 progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Genes Letais/genética , Genes de Plantas/genética , Hordeum/genética , Nitrito Redutases/genética , Nitritos/metabolismo , Apoproteínas/genética , Azidas/farmacologia , Western Blotting , Cruzamentos Genéticos , Genes Recessivos , Hordeum/efeitos dos fármacos , Mutagênese , Mutação , Nitrato Redutase (NAD(P)H) , Nitrato Redutases/biossíntese , Nitrito Redutases/imunologia , Seleção Genética , Azida Sódica , Distribuição TecidualRESUMO
Whether atrial natriuretic peptide (ANP)-evoked inhibition of corticotrophin-releasing factor (CRF)-stimulated ACTH secretion was also manifest in ACTH secreting AtT-20 pituitary tumour cells was investigated. ANP stimulated increases in cGMP accumulation at concentrations of the peptide above 10(-8) M which indicates the presence of the ANP receptors on these cells. CRF stimulated a concentration-dependent increase in ACTH secretion from AtT-20 cells which was unaffected by ANP, 8-bromo-cGMP, or sodium nitroprusside (SNP). Calcium stimulated a concentration-dependent increase in ACTH secretion from electrically permeabilised cells which was unaffected by co-incubation with cGMP but potentiated by cAMP. These results reveal the presence of ANP receptors on AtT-20 cells but suggest that an incomplete expression of the stimulus-secretion coupling mechanisms for ANP, at some point after cGMP production, prevents the effects of natriuretic peptides upon ACTH secretion being manifest in these cells.