RESUMEN
In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSA(KIT WT), expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into an SCF-independent clone, ROSA(KIT D816V), which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSA(KIT D816V) did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients.
Asunto(s)
Línea Celular , Mastocitos/patología , Mastocitosis Sistémica/genética , Proteínas Proto-Oncogénicas c-kit/genética , Animales , Western Blotting , Línea Celular/citología , Línea Celular/inmunología , Línea Celular/metabolismo , Separación Celular , Citometría de Flujo , Xenoinjertos , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , TransfecciónRESUMEN
Although the zeta-associated protein of 70 kDa (ZAP-70) is overexpressed in patients with chronic lymphocytic leukemia (CLL) displaying unmutated IGVH genes and poor prognosis, a previous microarray study from our group identified overexpression of LPL and ADAM29 genes among unmutated and mutated CLL, respectively. To assess the prognostic value of these genes, we quantified their expression by real-time quantitative polymerase chain reaction (PCR) in a cohort of 127 patients with CLL and correlated this with clinical outcome, IGVH mutational status, and ZAP-70 protein expression. IGVH mutational status, ZAP-70, and the LPL and ADAM29 mRNA ratios (L/A ratio) were predictive of event-free survival for the whole cohort and for patients with stage A disease. In patients in stage B and C, the L/A ratio was an independent prognostic factor, whereas ZAP-70 did not predict survival. Simultaneous usage of the L/A ratio and ZAP-70 expression allowed an almost perfect (99%) assessment of the IGVH status in the 80% of patients with concordant results (L/A+, ZAP-70+ or L/A-, ZAP-70-). LPL and ADAM29 gene expression could also be determined by a simple competitive multiplex reverse transcription PCR assay. Overall, quantification of LPL and ADAM29 gene expression is a strong prognostic indicator in CLL, providing better prognostic assessment than ZAP-70 in advanced stages of the disease.