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1.
J Hematol Oncol ; 6: 10, 2013 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-23343252

RESUMEN

BACKGROUND: We investigated the utility of bioluminescence imaging (BLI) using firefly luciferase in monoclonal and polyclonal populations of leukemia cells in vitro and in vivo. METHODS: Monoclonal and polyclonal human lymphoid and myeloid leukemia cell lines transduced with firefly luciferase were used for BLI. RESULTS: Kinetics and dynamics of bioluminescence signal were cell line dependent. Luciferase expression decreased significantly over time in polyclonal leukemia cells in vitro. Transplantation of polyclonal luciferase-tagged cells in mice resulted in inconsistent signal intensity. After selection of monoclonal cell populations, luciferase activity was stable, equal kinetic and dynamic of bioluminescence intensity and strong correlation between cell number and light emission in vitro were observed. We obtained an equal development of leukemia burden detected by luciferase activity in NOD-scid-gamma mice after transplantation of monoclonal populations. CONCLUSION: The use of monoclonal leukemia cells selected for stable and equal luciferase activity is recommended for experiments in vitro and xenograft mouse models. The findings are highly significant for bioluminescence imaging focused on pre-clinical drug development.


Asunto(s)
Modelos Animales de Enfermedad , Leucemia Experimental/patología , Luciferasas de Luciérnaga/metabolismo , Mediciones Luminiscentes , Fotones , Animales , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Subunidad gamma Común de Receptores de Interleucina/fisiología , Cinética , Leucemia Experimental/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas
2.
Mol Cancer Ther ; 12(11): 2367-77, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23997116

RESUMEN

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Although survival rates have improved, patients with certain biologic subtypes still have suboptimal outcomes. Current chemotherapeutic regimens are associated with short- and long-term toxicities and novel, less toxic therapeutic strategies are needed. Mer receptor tyrosine kinase is ectopically expressed in ALL patient samples and cell lines. Inhibition of Mer expression reduces prosurvival signaling, increases chemosensitivity, and delays development of leukemia in vivo, suggesting that Mer tyrosine kinase inhibitors are excellent candidates for targeted therapies. Brain and spinal tumors are the second most common malignancies in childhood. Multiple chemotherapy approaches and radiotherapies have been attempted, yet overall survival remains dismal. Mer is also abnormally expressed in atypical teratoid/rhabdoid tumors (AT/RT), providing a rationale for targeting Mer as a therapeutic strategy. We have previously described UNC569, the first small-molecule Mer inhibitor. This article describes the biochemical and biologic effects of UNC569 in ALL and AT/RT. UNC569 inhibited Mer activation and downstream signaling through ERK1/2 and AKT, determined by Western blot analysis. Treatment with UNC569 reduced proliferation/survival in liquid culture, decreased colony formation in methylcellulose/soft agar, and increased sensitivity to cytotoxic chemotherapies. MYC transgenic zebrafish with T-ALL were treated with UNC569 (4 µmol/L for two weeks). Fluorescence was quantified as indicator of the distribution of lymphoblasts, which express Mer and enhanced GFP. UNC569 induced more than 50% reduction in tumor burden compared with vehicle- and mock-treated fish. These data support further development of Mer inhibitors as effective therapies in ALL and AT/RT.


Asunto(s)
Antineoplásicos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Tumor Rabdoide/metabolismo , Teratoma/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Células Jurkat , Terapia Molecular Dirigida , Neoplasias Experimentales , Fosforilación/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Tumor Rabdoide/tratamiento farmacológico , Tumor Rabdoide/patología , Teratoma/tratamiento farmacológico , Teratoma/patología , Pez Cebra , Tirosina Quinasa c-Mer
3.
J Clin Invest ; 123(5): 2257-67, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23585477

RESUMEN

Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Melanoma/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Neoplasias Cutáneas/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Melanocitos/metabolismo , Ratones , Ratones SCID , Microscopía Fluorescente , Modelos Biológicos , Mutación , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Proto-Oncogenes Mas , Tirosina Quinasa c-Mer
4.
PLoS One ; 7(2): e31635, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22363695

RESUMEN

Mer tyrosine kinase is ectopically expressed in acute lymphoblastic leukemia and associated with enhanced chemoresistance and disease progression. While such effects are generally ascribed to increased engagement of oncogenic pathways downstream of Mer stimulation by its ligand, Gas6, Mer has not been characterized beyond the scope of its signaling activity. The present study explores Mer behavior following prolonged exposure to Gas6, a context similar to the Gas6-enriched microenvironment of the bone marrow, where a steady supply of ligand facilitates continuous engagement of Mer and likely sustains the presence of leukemic cells. Long-term Gas6 exposure induced production of a partially N-glycosylated form of Mer from newly synthesized stores of protein. Preferential expression of the partial Mer glycoform was associated with diminished levels of Mer on the cell surface and altered Mer localization within the nuclear-soluble and chromatin-bound fractions. The presence of Mer in the nucleus is a novel finding for this receptor, and the glycoform-specific preferences observed in each nuclear compartment suggest that glycosylation may influence Mer function within particular subcellular locales. Previous studies have established Mer as an attractive cancer biologic target, and understanding the complexity of its activity has important implications for potential strategies of Mer inhibition in leukemia therapy. Our results identify several novel features of Mer that expand the breadth of its functions and impact the development of therapeutic modalities designed to target Mer.


Asunto(s)
Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Leucemia/enzimología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Secuencia de Aminoácidos , Compartimento Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/enzimología , Secuencia Conservada , Glicosilación/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Datos de Secuencia Molecular , Peso Molecular , Señales de Exportación Nuclear , Señales de Localización Nuclear/química , Biosíntesis de Proteínas/efectos de los fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Tirosina Quinasas Receptoras/química , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Tirosina Quinasa c-Mer
5.
Protein Sci ; 18(4): 811-24, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19319933

RESUMEN

With the recent advances in NMR relaxation techniques, protein motions on functionally important timescales can be studied at atomic resolution. Here, we have used NMR-based relaxation experiments at several temperatures and both 600 and 900 MHz to characterize the inherent dynamics of the enzyme cyclophilin-A (CypA). We have discovered multiple chemical exchange processes within the enzyme that form a "dynamic continuum" that spans 20-30 A comprising active site residues and residues proximal to the active site. By combining mutagenesis with these NMR relaxation techniques, a simple method of counting the dynamically sampled conformations has been developed. Surprisingly, a combination of point mutations has allowed for the specific regulation of many of the exchange processes that occur within CypA, suggesting that the dynamics of an enzyme may be engineered.


Asunto(s)
Ciclofilina A/química , Ciclofilina A/genética , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Mutación Puntual , Conformación Proteica , Ingeniería de Proteínas , Electricidad Estática , Temperatura
6.
J Mol Biol ; 391(3): 518-35, 2009 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-19500591

RESUMEN

The CD147 receptor plays an integral role in numerous diseases by stimulating the expression of several protein families and serving as the receptor for extracellular cyclophilins; however, neither CD147 nor its interactions with its cyclophilin ligands have been well characterized in solution. CD147 is a unique protein in that it can function both at the cell membrane and after being released from cells where it continues to retain activity. Thus, the CD147 receptor functions through at least two mechanisms that include both cyclophilin-independent and cyclophilin-dependent modes of action. In regard to CD147 cyclophilin-independent activity, CD147 homophilic interactions are thought to underlie its activity. In regard to CD147 cyclophilin-dependent activity, cyclophilin/CD147 interactions may represent a novel means of signaling since cyclophilins are also peptidyl-prolyl isomerases. However, direct evidence of catalysis has not been shown within the cyclophilin/CD147 complex. In this report, we have characterized the solution behavior of the two most prevalent CD147 extracellular isoforms through biochemical methods that include gel-filtration and native gel analysis as well as directly through multiple NMR methods. All methods indicate that the extracellular immunoglobulin-like domains are monomeric in solution and, thus, suggest that CD147 homophilic interactions in vivo are mediated through other partners. Additionally, using multiple NMR techniques, we have identified and characterized the cyclophilin target site on CD147 and have shown for the first time that CD147 is also a substrate of its primary cyclophilin enzyme ligand, cyclophilin A.


Asunto(s)
Basigina/metabolismo , Ciclofilina A/metabolismo , Pliegue de Proteína , Basigina/química , Catálisis , Ciclofilina A/química , Humanos , Modelos Moleculares , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sindecano-1/metabolismo
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