RESUMEN
INTRODUCTION: Antimicrobial peptides (AMPs) have historically been evaluated for their role in protecting against uropathogens. However, there is mounting evidence to support their expression in noninfectious injury, with unclear meaning as to their function. It is possible that AMPs represent urothelial injury. Urinary tract obstruction is known to alter the urothelium; however, AMPs have not been evaluated for expression in this noninfectious injury. OBJECTIVE: A pilot study to compare urinary AMP expression in children undergoing surgical intervention for ureteropelvic junction obstruction (UPJO) with nonobstructed controls. STUDY DESIGN: Bladder urine was collected from consenting/assenting pediatric patients with UPJO at intervention. Control bladder urines were obtained from age-matched and sex-matched healthy children without known obstruction or infection. Enzyme-linked immunosorbent assays were run for the following AMPs: ß defense 1 (BD-1), neutrophil gelatinase-associated lipocalin (NGAL), cathelicidin (LL-37), hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), and human α defensin 5 (HD-5); and normalized to urine creatinine. Results were analyzed with Student's t-test or Mann-Whitney U test, when appropriate, and receiver operating characteristic curves. A P-value of <0.05 was considered significant. RESULTS: Thirty bladder urine samples were obtained from children with UPJO at the time of decompressive intervention. Mean patient age was 4.7 years (range 0.3-18.4); 20 (67%) patients were male. Fifteen bladder urine samples were obtained from age-matched and sex-matched controls. Urinary AMP levels were significantly higher in UPJO patients than controls for BD-1 (P = 0.015), NGAL (P < 0.001), LL-37 (P < 0.001), and HIP/PAP (P = 0.046). Optimal threshold values of these AMPs were determined, with each demonstrating significant odds ratios of predicting urinary obstruction. DISCUSSION: Certain urinary AMPs are altered even in noninfectious urinary tract pathology. This represents a novel induction of AMP expression, as the current study is the first to report elevations in BD-1 and HIP/PAP in urinary tract obstruction. This suggests other roles for these AMPs outside of their antimicrobial properties, and likely is a reflection of the urothelial and tubular stress resulting from obstructive uropathy. CONCLUSIONS: Induction of AMPs BD-1, NGAL, LL-37, and HIP/PAP was found to occur in urinary tract obstruction. Further evaluation of AMP expression as a biomarker of uroepithelial injury outside of infection is indicated.
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Péptidos Catiónicos Antimicrobianos/orina , Obstrucción Ureteral/orina , Urotelio/metabolismo , Adolescente , Biomarcadores/orina , Niño , Preescolar , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Proyectos Piloto , Obstrucción Ureteral/diagnóstico , Urinálisis , Adulto JovenRESUMEN
The FLT3 gene is mutated by an internal tandem duplication (ITD) in 20-25% of adults with acute myeloid leukemia (AML). We studied 82 adults <60 years of age with primary AML and normal cytogenetics, who received uniform high-dose therapy and found FLT3 ITD in 23 (28%) patients. When the 23 FLT3 ITD+ cases were compared with the 59 cases with wild-type (WT) FLT3, disease-free survival (DFS) was inferior (P = 0.03), yet overall survival (OS) was not different (P = 0.14). However, 8 (35%) of 23 FLT3 ITD/+ cases also lacked a FLT3 WT allele (FLT3(ITD-R)) as determined by PCR and loss of heterozygosity. Thus, three genotypic groups were identified: normal FLT3(WT/WT), heterozygous FLT3(ITD/WT), and hemizygous FLT3(ITD/-). DFS and OS were significantly inferior for patients with FLT3(ITD/-) (P = 0.0017 and P = 0.0014, respectively). Although DFS and OS for FLT3(WT/WT) and FLT3(ITD/WT) groups did not differ (P = 0.32 and P = 0.98, respectively), OS of the FLT3(ITD/-) group was worse than the FLT3(WT/WT) (P = 0.0005) and FLT3(ITD/WT) (P = 0.008) groups. We propose a model in which FLT3(ITD/-) represents a dominant positive, gain-of-function mutation providing AML cells with a greater growth advantage compared with cells having the FLT3(WT/WT) or FLT3(ITD/WT) genotypes. In conclusion, we have identified the FLT3(ITD/-) genotype as an adverse prognostic factor in de novo AML with normal cytogenetics. A poor prognosis of the relatively young FLT3(ITD/-) adults (median age, 37 years), despite treatment with current dose-intensive regimens, suggests that new treatment modalities, such as therapy with a FLT3 tyrosine kinase inhibitor, are clearly needed for this group of patients.
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Duplicación de Gen , Leucemia Mieloide/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Enfermedad Aguda , Adulto , Alelos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Supervivencia sin Enfermedad , Femenino , Humanos , Cariotipificación , Leucemia Mieloide/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Mutación , Secuencias Repetidas en Tándem , Resultado del Tratamiento , Tirosina Quinasa 3 Similar a fmsRESUMEN
Leukemia-associated Rho guanine nucleotide exchange factor (LARG) was originally identified as a fusion partner with mixed-lineage leukemia in a patient with acute myeloid leukemia. LARG possesses a tandem Dbl homology and pleckstrin homology domain structure and, consequently, may function as an activator of Rho GTPases. In this study, we demonstrate that LARG is a functional Dbl protein. Expression of LARG in cells caused activation of the serum response factor, a known downstream target of Rho-mediated signaling pathways. Transient overexpression of LARG did not activate the extracellular signal-regulated kinase or c-Jun NH(2)-terminal kinase mitogen-activated protein kinase cascade, suggesting LARG is not an activator of Ras, Rac, or Cdc42. We performed in vitro exchange assays where the isolated Dbl homology (DH) or DH/pleckstrin homology domains of LARG functioned as a strong activator of RhoA, but exhibited no activity toward Rac1 or Cdc42. We found that LARG could complex with RhoA, but not Rac or Cdc42, in vitro, and that expression of LARG caused an increase in the levels of the activated GTP-bound form of RhoA, but not Rac1 or Cdc42, in vivo. Thus, we conclude that LARG is a RhoA-specific guanine nucleotide exchange factor. Finally, like activated RhoA, we determined that LARG cooperated with activated Raf-1 to transform NIH3T3 cells. These data demonstrate that LARG is the first functional Dbl protein mutated in cancer and indicate LARG-mediated activation of RhoA may play a role in the development of human leukemias.
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Transformación Celular Neoplásica , Factores de Intercambio de Guanina Nucleótido/fisiología , Leucemia Mieloide Aguda/fisiopatología , Proteína de Unión al GTP rhoA/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Masculino , Proteínas Nucleares/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Factor de Respuesta SéricaRESUMEN
Signaling pathways between cell surface receptors and the BCL-2 family of proteins regulate cell death. Survival factors induce the phosphorylation and inactivation of BAD, a proapoptotic member. Purification of BAD kinase(s) identified membrane-based cAMP-dependent protein kinase (PKA) as a BAD Ser-112 (S112) site-specific kinase. PKA-specific inhibitors blocked the IL-3-induced phosphorylation on S112 of endogenous BAD as well as mitochondria-based BAD S112 kinase activity. A blocking peptide that disrupts type II PKA holoenzyme association with A-kinase-anchoring proteins (AKAPs) also inhibited BAD phosphorylation and eliminated the BAD S112 kinase activity at mitochondria. Thus, the anchoring of PKA to mitochondria represents a focused subcellular kinase/substrate interaction that inactivates BAD at its target organelle in response to a survival factor.
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Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Mitocondrias/enzimología , Animales , Apoptosis , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Interleucina-3/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Proteína Letal Asociada a bclRESUMEN
We have identified a gene at 11q23, telomeric to MLL, that encodes a guanine nucleotide exchange factor (GEF). This gene is transcribed into a 9.5-kb mRNA containing a 4.6-kb ORF. By Northern analysis, it was found to be expressed in all human tissues examined including peripheral blood leukocytes, spleen, prostate, testis, ovary, small intestine, colon, and minimally in thymus. Analysis of the predicted protein sequence indicates that it has strong homology to several members of the family of Rho GEFs that includes such oncogenes as Dbl, Vav, Tiam, and Bcr. A patient with primary acute myeloid leukemia (AML) and a karyotype of 51,XY,+8,+19,+3mar was found to have the 5' end of MLL at exon 6 fused in-frame with the 3' end of almost the entire ORF of this gene, which we named LARG for leukemia-associated Rho GEF. Transcriptional orientation of both genes at 11q23 is from centromere to telomere, consistent with other data that suggest the MLL-LARG fusion resulted from an interstitial deletion rather than a balanced translocation. LARG does not appear to have any homology with other MLL partner genes reported thus far. Thus, LARG represents an additional member of the GEF family and a novel MLL fusion partner in acute myeloid leukemia.