RESUMO
Aberrant DNA promoter methylation with associated gene silencing is a common epigenetic abnormality in acute lymphoblastic leukaemia (ALL) and is associated with poor survival. We have identified a family of transmembrane tyrosine phosphatase proteins as targets of hypermethylation in ALL and high-grade B cell lymphoma and demonstrated that this abnormal methylation correlates with transcript expression. PTPRG was methylated in 63% of ALL samples, PTPRK in 47%, PTPRM in 64% and PTPRO in 54% of cases, with most ALL samples containing methylation at multiple phosphatase loci. PTPRK promoter methylation was associated with a decreased overall survival in the cohort. Restoration of PTPRK transcript levels in leukaemia cells, where phosphatase transcript was silenced, reduced cell proliferation, inhibited colony formation and increased sensitivity to cytotoxic chemotherapy. These biological changes were associated with a reduction in levels of phosphorylated Erk1/2, Akt, STAT3 and STAT5 suggesting functional phosphatase activity after transcript re-expression. Methylation of the phosphatase promoters was reversible with decitabine and a histone deacetylase inhibitor, suggesting that PTPRK-mediated cell signalling pathways may be targeted with epigenetic therapies in lymphoid malignancy.
Assuntos
Metilação de DNA , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Tirosina Fosfatases/genética , Adolescente , Adulto , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Ilhas de CpG , Humanos , Janus Quinase 1/genética , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Modelos de Riscos Proporcionais , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Etoposídeo/administração & dosagem , Genes p53 , Imidazóis/administração & dosagem , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Piperazinas/administração & dosagem , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Proteínas Mutadas de Ataxia Telangiectasia , Inibidor de Quinase Dependente de Ciclina p21/genética , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológicoRESUMO
BACKGROUND: The clinical course of chronic lymphocytic leukaemia (CLL) is variable. ZAP-70 expression is believed to provide prognostic information. The flow cytometric detection of ZAP-70 is difficult because it is an intracellular antigen with weak expression in CLL. Consensus has not been reached as to the best method for measurement. METHODS: We analyzed 72 CLL patient samples for ZAP-70 expression and IgVH mutational status. Sensitivity and specificity of ZAP-70 expression against IgVH mutational status were assessed for two clones (2F3.2 and 1E7.2) and for four methods of analysis: percentage positivity (PP), comparing test to isotype control, ratio of geometric means of test and isotype control, and percentage and ratiometric methods comparing test and T/NK cell populations. The effects of anticoagulant, collection times, and time to analysis were also evaluated. RESULTS: Sensitivity and specificity were 85 and 88%, respectively, for Upstate PP; 70 and 88% for Caltag PP; 89 and 91% for Upstate ratio; 89 and 88% for Caltag ratio. Intraobserver variability was smaller when ZAP-70 expression was assessed using a ratiometric approach rather than the percentage method. By 48 h, we observed an average decrease of 13% in the Caltag ratio in the heparin preserved samples compared to an increase of 3% in those collected in EDTA. Within the first 24-h period, a greater percent variability was observed in those samples collected into EDTA compared with heparin. CONCLUSION: Our data support a rapid method for ZAP-70 measurement using commercially available fixation/permeabilization reagents, a conjugated antibody, and a ratiometric method of analysis that minimizes subjective interpretation of the results. This is a method of ZAP-70 assessment that could be included in a routine diagnostic CLL panel; however, the choice of anticoagulant and time of analysis after collection are critical factors in accurate assessment of ZAP-70 expression.
Assuntos
Anticorpos/imunologia , Anticoagulantes/farmacologia , Citometria de Fluxo/métodos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Proteína-Tirosina Quinase ZAP-70/análise , Reações Antígeno-Anticorpo , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/química , Biomarcadores Tumorais/imunologia , Progressão da Doença , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Pessoa de Meia-Idade , Mutação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem , Fatores de Tempo , Proteína-Tirosina Quinase ZAP-70/efeitos dos fármacos , Proteína-Tirosina Quinase ZAP-70/imunologiaRESUMO
Monolayer cultures of rat fetal distal lung epithelial (FDLE) cells generated larger spontaneous short circuit currents (ISC) when maintained (48 h) at neonatal alveolar PO2 (100 mmHg) than at fetal PO2 (23 mmHg). When cells were shifted between these atmospheres in order to impose a rise in PO2 equivalent to that seen at birth, no rise in ISC was seen after 6 h but the response was fully established by 24 h. Studies of basolaterally permeabilised cells revealed a small rise in apical Na+ conductance (GNa) 6 h after PO2 was raised but no further change had occurred by 24 h. A substantial rise was, however, seen after 48 h. Reporter gene assays showed that no activation of the -ENaC (epithelial Na+ channel -subunit) promoter was discernible 24 h after PO2 was raised but increased transcriptional activity was seen at 48 h. Studies of apically permeabilised cells showed that a small rise in Na+ pump capacity was evident 6 h after PO2 was raised and, in common with the rise in ISC, this effect was fully established by 24 h. The rise in ISC thus develops 6-24 h after PO2 is raised and is due, primarily, to increased Na+ pump capacity. The increase in GNa thus coincides with activation of the -ENaC promoter but these effects occur after the rise in ISC is fully established and so cannot underlie this physiological response. The increased transcription may be an adaptation to increased Na+ transport and not its cause.
