RESUMEN
The carboxy-terminal truncated p53 alternative spliced isoforms, p53ß and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53(null) background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53ß and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53ß and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1)), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53ß protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53ß and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53ß and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.
Asunto(s)
Antineoplásicos/farmacología , Eliminación de Gen , Neoplasias Pulmonares/patología , Osteosarcoma/patología , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica , Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Ratones , Osteosarcoma/genética , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genéticaRESUMEN
The ability to visualize reporter gene expression in vivo has revolutionized all facets of biologic investigation and none more so than imaging applications in oncology. Near-infrared reporter gene imaging may facilitate more accurate evaluation of chemotherapeutic response in preclinical models of orthotopic and metastatic cancers. We report the development of a cell permeable, quenched squarine probe (CytoCy5S), which is reduced by Escherichia coli nitroreductase (NTR), resulting in a near-infrared fluorescent product. Time-domain molecular imaging of NTR/CytoCy5S reporter platform permitted noninvasive monitoring of disease progression in orthotopic xenografts of disseminated leukemia, lung, and metastatic breast cancer. This methodology facilitated therapeutic evaluation of NTR gene-directed enzymatic prodrug therapy with conventional metronidazole antibiotics. These studies show NTR/CytoCy5S as a near-infrared gene reporter system with broad preclinical and prospective clinical applications within imaging, and gene therapy, of cancer.
Asunto(s)
Carbocianinas/metabolismo , Neoplasias/metabolismo , Nitrorreductasas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antiinfecciosos/farmacología , Carbocianinas/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Imagen por Resonancia Magnética/métodos , Metronidazol/metabolismo , Metronidazol/farmacología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microscopía Fluorescente/métodos , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Nitroimidazoles/metabolismo , Nitroimidazoles/farmacología , Nitrorreductasas/química , Nitrorreductasas/genética , Reproducibilidad de los Resultados , TransfecciónRESUMEN
Human xenografts of acute myeloid leukemia (AML) in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice result in disease states of diffuse, nonpalpable tissue infiltrates exhibiting a variable disease course, with some animals not developing a disease phenotype. Thus, disease staging and, more critically, quantification of preclinical therapeutic effect in these models are particularly difficult. In this study, we present the generation of a green fluorescent protein (GFP)-labeled human leukemic cell line, NB4, and validate the potential of a time-domain imager fitted with a 470 nm picosecond pulsed laser diode to decouple GFP fluorescence from autofluorescence on the basis of fluorescence lifetime and thus determine the depth and relative concentration of GFP inclusions in phantoms of homogeneous and heterogeneous optical properties. Subsequently, we developed an optical imageable human xenograft model of NB4-GFP AML and illustrate early disease detection, depth discrimination of leukemic infiltrates, and longitudinal monitoring of disease course employing time-domain optical imaging. We conclude that early disease detection through use of time-domain imaging in this initially slowly progressing AML xenograft model permits accurate disease staging and should aid in future preclinical development of therapeutics for AML.