RESUMEN
Gene amplification represents one of the molecular mechanisms of oncogene overexpression in many types of tumors. Homogeneously staining regions (HSRs) are cytogenetic hallmarks of gene amplification. Rhabdomyosarcoma is the most common malignant soft-tissue tumor in children. RMS-YM is an embryonal rhabdomyosarcoma cell line that possesses 3 HSRs. This cytogenetic finding suggests the presence of gene amplifications associated with tumor development or progression in RMS-YM. Here, using fluorescence in situ hybridization, we detected high amplification of the MDM2 gene in the HSRs of RMS-YM. We also refined the region of the amplicon and identified that the FRS2 gene and others are amplified in RMS-YM. MDM2 and FRS2 play important roles as a regulator of p53 and a mediator of FGF signaling, respectively, and thus are potential molecular targets for therapy in many different tumors. RMS-YM may be useful for studies of the molecular pathways of tumorigenesis and tumor progression in rhabdomyosarcoma and for in vitro evaluation of newly developed therapeutic agents that target MDM2 or FRS2.
Asunto(s)
Cromosomas Humanos Par 12/genética , Amplificación de Genes/genética , Oncogenes , Proteínas Proto-Oncogénicas c-mdm2/genética , Rabdomiosarcoma Embrionario/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Preescolar , Aberraciones Cromosómicas , Bandeo Cromosómico , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , Transducción de Señal/genéticaRESUMEN
We report on an 11-year-old girl who developed steroid-resistant nephrotic syndrome (NS) at the onset of systemic lupus erythematosus (SLE), and clinical and renal histological findings suggested that her NS would be associated with SLE-related podocytopathy. Although initial treatment with intravenous pulse methylprednisolone was ineffective, following treatment with cyclosporine and an angiotensin receptor blocker was effective for her nephrotic proteinuria. She had developed posterior reversible encephalopathy syndrome (PRES), and mycophenolate mofetil (MMF) was started instead of cyclosporine. At present, 45 months after the onset, she is in remission of both NS and SLE. This case indicates that NS associated with SLE-related podocytopathy should be included in the spectrum of glomerulopathy accompanying SLE, also in the pediatric population.
Asunto(s)
Glucocorticoides/uso terapéutico , Lupus Eritematoso Sistémico/complicaciones , Metilprednisolona/uso terapéutico , Síndrome Nefrótico/tratamiento farmacológico , Síndrome Nefrótico/etiología , Niño , Ciclosporina , Resistencia a Medicamentos , Femenino , HumanosRESUMEN
BACKGROUND: Inflammatory mediators may have decisive roles at different stages of tumour development. Mediators within the pentraxin family may be used as strong biomarkers in prognosis of advanced pancreatic carcinoma patients. METHODS: Using pancreatic carcinoma cell lines and gene transfectant, we measured long pentraxin (PTX3) level in culture solution and carried out cellular migration assay in vitro. In vivo study of the treatment-naive patients with advanced pancreatic carcinoma assigned to undergo gemcitabine therapy was prospectively conducted to measure and investigate the role of plasma PTX3, C-reactive protein (CRP), and eight inflammatory mediators by using collected clinical data. RESULTS: Elevated PTX3 production was observed in several cell lines, and a direct relationship between migratory activity and PTX3 level was identified in vitro. High PTX3 level (117 days) was significantly less than that of patients with low PTX3 level (357 days, P<0.001). Multivariate analysis of the pancreatic carcinoma revealed a strong correlation between pentraxin family member expression and prognosis of pancreatic carcinoma. The relationship between PTX3 expression and the expression of other pro-inflammatory mediators indicated that PTX3 level is positively correlated with levels of CRP, interleukin-6, and macrophage-inhibitory factor. CONCLUSION: Pentraxin family members, especially PTX3, may be used as promising biomarkers in the prognosis of pancreatic carcinoma patients.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Proteína C-Reactiva/biosíntesis , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Componente Amiloide P Sérico/biosíntesis , Adulto , Anciano , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/sangre , Línea Celular Tumoral , Movimiento Celular/fisiología , Desoxicitidina/uso terapéutico , Femenino , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Pronóstico , Estudios Prospectivos , Transfección , Resultado del Tratamiento , GemcitabinaRESUMEN
BACKGROUND: Scabies is a contagious dermatosis. The risk factors for its transmission remain unclear. A scabies outbreak, involving patients who were receiving chemotherapy for haematological malignancies, occurred at our hospital. METHODS: The outbreak population was analysed to determine whether the incidence of scabies was higher among contact patients receiving chemotherapy for haematological malignancies. RESULTS: A patient with crusted scabies was the index case, and 18 of 78 contact healthcare workers (HCWs) and 22 of 135 contact patients were diagnosed with classical scabies. Ten of 17 contact patients with haematological malignancies and 12 of 118 contact patients with other diseases were infected with scabies. The incidence rate was significantly higher among the patients with haematological malignancies (P<0.001). The patients with haematological malignancies had a significantly lower mean minimum neutrophil count than those with other diseases (1159/µL vs 3761/µL, P=0.0012). Most haematological patients did not require special nursing assistance, suggesting that the higher incidence of scabies among these patients resulted from their immunodeficiency rather than greater skin-to-skin contact with infected HCWs. CONCLUSION: Our study suggests that patients receiving chemotherapy for haematological malignancies are more susceptible to scabies than patients with other diseases, and require stricter protection.
Asunto(s)
Susceptibilidad a Enfermedades/inducido químicamente , Quimioterapia , Neoplasias Hematológicas/complicaciones , Neoplasias Hematológicas/tratamiento farmacológico , Escabiosis/etiología , Anciano , Anciano de 80 o más Años , Infección Hospitalaria/epidemiología , Infección Hospitalaria/prevención & control , Brotes de Enfermedades/estadística & datos numéricos , Susceptibilidad a Enfermedades/parasitología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Femenino , Personal de Salud/estadística & datos numéricos , Hospitales/estadística & datos numéricos , Humanos , Control de Infecciones/métodos , Transmisión de Enfermedad Infecciosa de Paciente a Profesional/estadística & datos numéricos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Escabiosis/epidemiología , Escabiosis/transmisiónRESUMEN
The tumour suppressor gene RASSF1A is known to be frequently silenced by promoter hypermethylation in neuroblastoma tumours. Here we explored the possible prognostic significance of aberrant promoter hypermethylation of RASSF1A in serum DNA samples of patients with neuroblastoma as a surrogate marker for circulating tumour cells. We analysed the methylation status of the RASSF1A gene in matched tumour and pretreatment serum DNA obtained from 68 neuroblastoma patients. Hypermethylation of RASSF1A in tumour samples was found in 64 patients (94%). In contrast, serum methylation of RASSF1A was observed in 17 patients (25%). Serum methylation of RASSF1A was found to be statistically associated with age > or =12 months at diagnosis (P=0.002), stage 4 (P<0.001) and MYCN amplification (P<0.001). The influence of serum RASSF1A methylation on prognosis was found to be comparable with that of the currently most reliable marker, MYCN amplification on univariate analysis (hazard ratio, 9.2; 95% confidence interval (CI), 2.8-30.1; P<0.001). In multivariate analysis of survival, methylation of RASSF1A in serum had a hazard ratio of 2.4 (95% CI, 0.6-9.2), although this association did not reach statistical significance (P=0.194). These findings show that the methylation status of RASSF1A in the serum of patients with neuroblastoma has the potential to become a prognostic predictor of outcome.
Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , ADN/sangre , Neuroblastoma/sangre , Proteínas Supresoras de Tumor/genética , Femenino , Humanos , Lactante , Masculino , Proteína Proto-Oncogénica N-Myc , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Pronóstico , Regiones Promotoras Genéticas/genética , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
The immunosuppressant rapamycin interferes with G1-phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E-binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G1-phase progression in mammalian cells.
Asunto(s)
Fosfoproteínas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Polienos/farmacología , Proteínas Quinasas , Proteínas Represoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Androstadienos/farmacología , Animales , Proteínas Portadoras/farmacología , Proteínas de Ciclo Celular , Línea Celular , Proteínas de Unión al ADN/farmacología , Factor 4E Eucariótico de Iniciación , Fase G1 , Proteínas de Choque Térmico/farmacología , Humanos , Insulina/farmacología , Péptidos y Proteínas de Señalización Intracelular , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/genética , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Ratas , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Transducción de Señal , Sirolimus , Serina-Treonina Quinasas TOR , Proteínas de Unión a Tacrolimus , Transfección , Células Tumorales Cultivadas , WortmaninaAsunto(s)
Anticonvulsivantes/uso terapéutico , Epilepsia/tratamiento farmacológico , Proteínas Munc18/genética , Mutación/genética , Piracetam/análogos & derivados , Epilepsia/diagnóstico , Epilepsia/genética , Humanos , Lactante , Levetiracetam , Masculino , Piracetam/uso terapéutico , Síndrome , Resultado del TratamientoRESUMEN
Two distinct signaling pathways regulate the survival of interleukin-3 (IL-3)-dependent hematopoietic progenitors. One originates from the membrane-proximal portion of the cytoplasmic domain of the IL-3 receptor (betac chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor and is involved in the regulation of Bcl-x(L) through activation of STAT5. The other pathway emanates from the distal region of the betac chain and overlaps with downstream signals from constitutively active Ras proteins. Although the latter pathway is indispensable for cell survival, its downstream targets remain largely undefined. Here we show that the expression of Bim, a member of the BH3-only subfamily of cell death activators, is downregulated by IL-3 signaling through either of two major Ras pathways: Raf/mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/mammalian target of rapamycin. Akt/phosphokinase B does not appear to play a significant role in this regulatory cascade. Bim downregulation has important implications for cell survival, since enforced expression of this death activator at levels equivalent to those induced by cytokine withdrawal led to apoptosis even in the presence of IL-3. We conclude that Bim is a pivotal molecule in cytokine regulation of hematopoietic cell survival.
Asunto(s)
Proteínas Portadoras/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de la Membrana , Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis , Proteína 11 Similar a Bcl2 , Línea Celular , Supervivencia Celular , Regulación hacia Abajo , Humanos , Interleucina-3/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Serina-Treonina Quinasas TOR , Proteínas ras/metabolismoRESUMEN
In normal development the neural crest gives rise to sympathetic neuroblasts, sensory and autonomic ganglia, as well as Schwann cells. One tumor arising from this tissue is the neuroblastoma (NB), a malignancy of the adrenergic component of the sympathetic nervous system. Recent histological studies have shown that neuroblastomas can present with a schwannian cell component, rich in S100 protein. We have investigated the differentiation of NB cell lines, GOTO and RT-LN-1, into a schwannian cell phenotype using bromodeoxyuridine (BrdU). This agent induced morphological changes in these cell lines. Flat-epithelial cells were identified in the GOTO cell line and both flat-epithelial and neuronal phenotypes were found in the RT-LN-1 cell line. S100 protein (beta-Subunit) was induced in both cell lines after 18-25 days of BrdU treatment as determined by enzyme-linked immunoassay. In addition increase in the beta-subunit of S100 protein was identified in BrdU-treated flat-epithelial cells by indirect immunofluorescence using a monoclonal antibody specific for the beta-subunit of the protein. Cyclic nucleotide phosphodiesterase activity significantly increased in both BrdU-treated NB cell lines, as compared with nontreated cells. However no significant increase of glial fibrillary acidic protein in BrdU-treated cells was found either by enzyme-linked immunoassay or indirect immunofluorescence using a monoclonal antibody to glial fibrillary acidic protein. Thus, cells with Schwann cell characteristics can clearly be identified in the neuroblastoma cell lines after BrdU treatment. Fluorescence-activated cell sorting analysis revealed no quantitative changes in cell membrane antigens recognized by monoclonal antibodies UJ-13A (neuroectodermal associated antigen) and anti-Thy-1 (Thy-1) on BrdU treatment. In contrast, UJ-127-11 (neuroectodermal associated) decreased, and W6/32 and BB7.7 (HLA-ABC) and BBM.1 (beta 2-microglobulin) markedly increased in both BrdU-treated cell lines. No induction of L243 (HLA-DR), B7/21 (HLA-DP), and Genox 3.55 (HLA-DQ) was noted. The increased HLA-ABC (HLA class I) antigen may enable BrdU-treated NB cells to be recognized by cytotoxic T-cells. This may be related to the pathological evidence that NB patients whose tumors are rich in S100 protein have a better prognosis. Further studies on the potential of differentiation agents to induce a phenotypic change, that is associated with an improved prognosis for NB patients, are required.
Asunto(s)
Neuroblastoma/patología , Células de Schwann/patología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/análisis , Antígenos de Superficie/análisis , Bromodesoxiuridina/farmacología , Diferenciación Celular/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/análisis , Humanos , Filamentos Intermedios/inmunología , Neuroblastoma/análisis , Proteínas S100/análisis , Células Tumorales CultivadasRESUMEN
The relationship between G(1) checkpoint function and rapamycininduced apoptosis was examined using two human rhabdomyosarcoma cell lines, Rh1 and Rh30, that express mutated p53 alleles. Serum-starved tumor cells became apoptotic when exposed to rapamycin, but were completely protected by expression of a rapamycin-resistant mutant mTOR. Exposure to rapamycin (100 ng/ml) for 24 h significantly increased the proportion of Rh1 and Rh30 cells in G(1) phase, although there were no significant changes in expression of cyclins D1, E, or A in drug-treated cells. To determine whether apoptosis was associated with continued slow progression through G(1) to S phase, cells were exposed to rapamycin for 24 h, then labeled with bromodeoxyuridine (BrdUrd). Histochemical analysis showed that >90% of cells with morphological signs of apoptosis had incorporated BRDURD: To determine whether restoration of G(1) arrest could protect cells from rapamycin-induced apoptosis, cells were infected with replication-defective adenovirus expressing either p53 or p21(CIP1). Infection of Rh30 cells with either Ad-p53 or Ad-p21, but not control virus (Ad-beta-gal), induced G(1) accumulation, up-regulation of p21(CIP1), and complete protection of cells from rapamycin-induced apoptosis. Within 24 h of infection of Rh1 cells with Ad-p21, expression of cyclin A was reduced by >90%. Similar results were obtained after Ad-p53 infection of Rh30 cells. Consistent with these data, incorporation of [(3)H]thymidine or BrdUrd into DNA was significantly inhibited, as was cyclin-dependent kinase 2 activity. These data indicate that rapamycin-induced apoptosis in tumor cells is a consequence of continued G(1) progression during mTOR inhibition and that arresting cells in G(1) phase, by overexpression of p53 or p21(CIP1), protects against apoptosis. The response to rapamycin was next examined in wild-type or murine embryo fibroblasts nullizygous for p53or p21(CIP1). Under serum-free conditions, rapamycin-treated wild-type MEFs showed no increase in apoptosis compared to controls. In contrast, rapamycin significantly induced apoptosis in cells deficient in p53 ( approximately 2.4-fold) or p21(CIP1) ( approximately 5.5-fold). Infection of p53(-/-) MEFs with Ad-p53 or Ad-p21 completely protected against rapamycin-induced apoptosis. Under serum-containing conditions, rapamycin inhibited incorporation of BrdUrd significantly more in wild-type murine embryo fibroblasts (MEFs) than in those lacking p53 or p21(CIP1). When BrdUrd was added 24 h after rapamycin, almost 90% and 70% of cells lacking p53 or p21(CIP1), respectively, incorporated nucleoside. In contrast, only 19% of wild-type cells incorporated BrdUrd in the presence of rapamycin. Western blot analysis of cyclin levels showed that rapamycin had little effect on levels of cyclins D1 or E in any MEF strain. However, cyclin A was reduced to very low levels by rapamycin in wild-type cells, but remained high in cells lacking p53 or p21(CIP1). Taken together, the data suggest that p53 cooperates in enforcing G(1) cell cycle arrest, leading to a cytostatic response to rapamycin. In contrast, in tumor cells, or MEFs, having deficient p53 function the response to this agent may be cell cycle progression and apoptosis.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Ciclinas/fisiología , Fase G1/efectos de los fármacos , Proteínas Quinasas , Sirolimus/farmacología , Proteína p53 Supresora de Tumor/fisiología , Adenoviridae/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Ciclinas/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fase G1/fisiología , Humanos , Ratones , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Rabdomiosarcoma/tratamiento farmacológico , Rabdomiosarcoma/patología , Serina-Treonina Quinasas TOR , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Insulin-like growth factor I (IGF-I) stimulates proliferation, survival, and differentiation in many cell types, including pediatric neuroblastomas. The effect is mediated via the type I IGF-I receptor (IGF-IR), which is essential for growth in these cells. Several lines of evidence indicate that IGF-IR function may be particularly important in the pathogenesis of neuroblastoma. Amplification of the N-myc oncogene or overexpression of N-Myc oncoprotein has been reported to be associated with resistance to therapy and poor prognosis of neuroblastomas. It was therefore of interest to analyze whether IGF-I signaling regulated expression of N-myc in KP-N-RT human neuroblastoma cells as an experimental model that has amplified N-myc. We found that IGF-I induces N-myc mRNA and protein in the KP-N-RT with maximums of four and six times more than the basal level at 2 and 3 h after stimulation, respectively. These effects of IGF-I were blocked by a neutralizing antibody against IGF-IR (alpha-IR3). Exogenous IGF-I induced phosphorylation and activation of extracellular signal-regulated kinases p44/42 (ERK1 and ERK2), with a maximal level 30 min after the stimulation. The MEK1 inhibitor PD98059 reduced IGF-I-mediated p44/42 MAPKs phosphorylation and produced a parallel reduction of IGF-I-stimulated N-Myc induction. Furthermore, both alpha-IR3 and PD98059 inhibited G1-S cell cycle progression stimulated by IGF-I. Our results demonstrate that IGF-I induces N-Myc in the KP-N-RT neuroblastoma cell line at the RNA level and establishes a clear correlation between N-Myc induction and activation of p44/42 MAPK signaling.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Proteínas de Neoplasias/fisiología , Neuroblastoma/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Fase G1/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , ARN Mensajero/metabolismo , Fase S/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The mammalian target of rapamycin (mTOR) has been shown to link growth factor signaling and posttranscriptional control of translation of proteins that are frequently involved in cell cycle progression. However, the role of this pathway in cell survival has not been demonstrated. Here, we report that rapamycin, a specific inhibitor of mTOR kinase, induces G1 cell cycle arrest and apoptosis in two rhabdomyosarcoma cell lines (Rh1 and Rh30) under conditions of autocrine cell growth. To examine the kinetics of rapamycin action, we next determined the rapamycin sensitivity of rhabdomyosarcoma cells exposed briefly (1 h) or continuously (6 days). Results demonstrate that Rh1 and Rh30 cells were equally sensitive to rapamycin-induced growth arrest and apoptosis under either condition. Apoptosis was detected between 24 and 144 h of exposure to rapamycin. Both cell lines have mutant p53; hence, rapamycin-induced apoptosis appears to be a p53-independent process. To determine whether induction of apoptosis by rapamycin was specifically due to inhibition of mTOR signaling, we engineered Rh1 and Rh30 clones to stably express a mutant form of mTOR that was resistant to rapamycin (Ser2035-->Ile; designated mTOR-rr). Rh1 and Rh30 mTOR-rr clones were highly resistant (>3000-fold) to both growth inhibition and apoptosis induced by rapamycin. These results are the first to indicate that rapamycin-induced apoptosis is mediated by inhibition of mTOR. Exogenous insulin-like growth factor (IGF)-I protected both Rh1 and Rh30 from apoptosis, without reactivating ribosomal p70 S6 kinase (p70S6K) downstream of mTOR. However, in rapamycin-treated cultures, the response to IGF-I differed between the cell lines: Rh1 cells proliferated normally, whereas Rh30 cells remained arrested in G1 phase but viable. Rapamycin is known to inhibit synthesis of specific proteins but did not inhibit synthesis or alter the levels of mTOR. To examine the rate at which the mTOR pathway recovered, the ability of IGF-I to stimulate p70S6K activity was followed in cells treated for 1 h with rapamycin and then allowed to recover in medium containing > or =100-fold excess of FK506 (to prevent rapamycin from rebinding to its cytosolic receptor FKBP-12). Our results indicate that, in Rh1 cells, rapamycin dissociates relatively slowly from FKBP-12, with a t1/2 of approximately 17.5 h. in the presence of FK506, whereas there was no recovery of p70S6K activity in the absence of this competitor. This was of interest because rapamycin was relatively unstable under conditions of cell culture having a biological t1/2 of approximately 9.9 h. These results help to explain why cells are sensitive following short exposures to rapamycin and may be useful in guiding the use of rapamycin analogues that are entering clinical trials as novel antitumor agents.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Portadoras , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Rabdomiosarcoma/patología , Sirolimus/farmacología , Proteína p53 Supresora de Tumor/fisiología , Sitios de Unión , Fase G1/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Serina-Treonina Quinasas TOR , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy that primarily develops during infancy and early childhood. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is dismal; therefore, a greater understanding of the biology of this disease is required to establish novel therapies. In this study, we identified a highly tumorigenic sub-population in MRT, based on the expression of CD146 (also known as melanoma cell adhesion molecule), a cell adhesion molecule expressed by neural crest cells and various derivatives. CD146+ cells isolated from four MRT cell lines by cell sorting exhibited enhanced self-renewal and invasive potential in vitro. In a xenograft model using immunodeficient NOD/Shi-scid IL-2Rγ-null mice, purified CD146+ cells obtained from MRT cell lines or a primary tumor exhibited the exclusive ability to form tumors in vivo. Blocking of CD146-related mechanisms, either by short hairpin RNA knockdown or treatment with a polyclonal antibody against CD146, effectively suppressed tumor growth of MRT cells both in vitro and in vivo via induction of apoptosis by inactivating Akt. Furthermore, CD146 positivity in immunohistological analysis of 11 MRT patient samples was associated with poor patient outcomes. These results suggest that CD146 defines a distinct sub-population in MRT with high tumorigenic capacity and that this marker represents a promising therapeutic target.
Asunto(s)
Biomarcadores de Tumor/genética , Tumor Rabdoide/genética , Tumor Rabdoide/terapia , Adolescente , Adulto , Anciano , Animales , Apoptosis/genética , Biomarcadores de Tumor/biosíntesis , Antígeno CD146/biosíntesis , Antígeno CD146/genética , Carcinogénesis/genética , Linaje de la Célula/genética , Niño , Preescolar , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Ratones , Persona de Mediana Edad , Cresta Neural/patología , Tumor Rabdoide/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Curative radiotherapy is the first choice of therapy for T1 and T2 stage laryngeal squamous cell carcinoma (LSCC) patients to preserve their phonation. Patients with recurrent tumors who undergo salvage surgery require prolonged nasal feeding. Therefore, clinical interest has been focused on elucidating a predictive factor indicating which tumors are likely to be radiosensitive before radiotherapy. We analyzed the relations between radiosensitivity and clinicopathological factors (gender, tumor location, histological factors, and clinical tumor-node-metastasis stage), expression of apoptosis-related proteins (p53, bax, bcl-2), apoptotic index using the terminal deoxynucleotidyltransferase-mediated nick end labeling method, expression of cell proliferation-related proteins (Ki-67-labeling index and epidermal growth factor receptor overexpression) and microvessel density (MVD, vessels/field = 0.391 mm2) in biopsy specimens from 31 LSCC patients given radiotherapy (total radiotherapy dose of 52-70 Gy over 4-6.5 weeks). Univariate analysis revealed that tumors with a high MVD (> or =35 vessels/field) showed better radiosensitivity than those with a low MVD (<35 vessels/field, P = 0.008) and that a high Ki-67-labeling index (> or =40%) was weakly associated with radiosensitivity (P = 0.056). Multivariate analysis and Kaplan-Meier analysis showed that MVD alone had significant predictive power for radiosensitivity in T1 and T2 stage LSCCs after radiotherapy (P = 0.012, 0.0003, respectively). No significant association between clinicopathological factors, or of overexpression of p53, bax, bcl-2, epidermal growth factor receptor, or apoptotic index, with radiosensitivity was found. These results indicate that MVD is a potentially useful clinical factor predicting radiosensitivity for patients with early stage LSCCs before treatment.
Asunto(s)
Carcinoma de Células Escamosas/irrigación sanguínea , Carcinoma de Células Escamosas/radioterapia , Neoplasias Laríngeas/irrigación sanguínea , Neoplasias Laríngeas/radioterapia , Neovascularización Patológica/fisiopatología , Tolerancia a Radiación/fisiología , Anciano , Anciano de 80 o más Años , Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Apoptosis/fisiología , Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/biosíntesis , Femenino , Humanos , Antígeno Ki-67/biosíntesis , Neoplasias Laríngeas/metabolismo , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/biosíntesis , Proteína X Asociada a bcl-2RESUMEN
PURPOSE: To confirm that human cancer cells show p53-dependent heat sensitivity through an apoptosis-related mechanism, we examined the heat sensitivity and Bax-mediated apoptosis after heating in a human squamous cell carcinoma cell line, SAS, with identical genetic backgrounds except for the p53 status. MATERIALS AND METHODS: We performed colony formation assay, Western blotting and analyses of apoptosis, using the SAS cells transfected with pC53-248 vector with mutant p53 gene (SAS/Trp248 cells) or the cells transfected with pCMV-Neo-Bam vector (SAS/neo cells) as a control. RESULTS: SAS/Trp248 cells showed heat resistance due to the dominant negative nature of mp53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly after heating in SAS/neo cells, but increased very little in SAS/Trp248 cells. CONCLUSION: These results suggest that heat resistance brought by mp53-transfection is p53-dependent and closely correlates with the induction of apoptosis in human squamous cell carcinomas.
Asunto(s)
Apoptosis/fisiología , Carcinoma de Células Escamosas/fisiopatología , Genes p53/fisiología , Neoplasias de Cabeza y Cuello/fisiopatología , Hipertermia Inducida , Proteínas Proto-Oncogénicas c-bcl-2 , Apoptosis/genética , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/terapia , Caspasa 3 , Caspasas/metabolismo , Supervivencia Celular , Fragmentación del ADN , Activación Enzimática , Regulación de la Expresión Génica , Vectores Genéticos/genética , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/terapia , Humanos , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales Cultivadas , Ensayo de Tumor de Célula Madre , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2RESUMEN
The mitogenic substance P receptor (NK-1 subtype) is expressed in many primary human tumors with the highest frequency of expression appearing in astrocytomas and glioblastomas (75% and 100%, respectively). Recently, we showed that substance P neuropeptide induces DNA synthesis in the human astrocytoma U-373MG cells by activating the mitogen-activated protein (MAP) kinase pathway leading to the induction of c-Fos and c-Myc expression. The induction of these immediate early genes is necessary for the progression of cells form G1 to S phase of the cell cycle. In this study, we demonstrate that U-373MG cells are highly sensitive to the growth-inhibitory action of rapamycin at nanomolar concentrations (IC50 <1 ng/ml). We also show that SP peptide stimulates protein synthesis in the U-373MG cell line by activating a rapamycin-sensitive signaling pathway. Further, we demonstrate that SP is potent in stimulating PHAS-I protein (also known as 4E-BP1) phosphorylation and p70 S6 kinase (p70(S6K)) phosphorylation and enzymatic activity, and that this stimulation is inhibited by subnanomolar concentrations of rapamycin. In contrast, rapamycin was not at all effective in repressing SP-induced activation of MAP kinase pathway, c-Fos phosphoprotein expression, and DNA synthesis in U-373MG astrocytoma cells.
RESUMEN
We have investigated the CDDP sensitivities of two tongue cancer cell lines with differing p53 genetic status, one with wild-type p53 (SAS) and the other with mutant-type p53 (HSC-4). SAS was about 2 times more sensitive at the D10 dose and demonstrated increased p53 and Bax protein levels at 10 h after CDDP treatment on Western blot analysis. On the other hand, overexpression of p53 in HSC-4 was observed without CDDP treatment and no elevation of Bax could be detected. Apoptosis was observed after CDDP treatment in SAS but not in HSC-4 by Hoechst 33342-staining and electrophoresis methods. These findings indicate that p53 plays an important role in apoptosis as a positive regulator of Bax expression. It is suggested that p53 status may have predictive potential with regard to response to CDDP therapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/patología , Cisplatino/farmacología , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-bcl-2 , Neoplasias de la Lengua/patología , Proteína p53 Supresora de Tumor/fisiología , Carcinoma de Células Escamosas/genética , ADN de Neoplasias/genética , Resistencia a Antineoplásicos , Genes p53 , Humanos , Proteínas de Neoplasias/biosíntesis , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Proteínas Proto-Oncogénicas/biosíntesis , Neoplasias de la Lengua/genética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patología , Proteína p53 Supresora de Tumor/biosíntesis , Proteína X Asociada a bcl-2RESUMEN
The task-dependent laterality of the auditory cortices was investigated by measuring the magnetic fields elicited by three forms of a Japanese verb, which differed in terms of prosodic and phonetic cues. Significant task-dependent magnetic fields were found in both hemispheres during a prosody-related task, but only in the left during a phoneme-related task. The latency was similar to the mismatch negatively which reflects the neural activity of automatic cue decoding. These results suggest that task-dependent schemata are activated at least partially in parallel with automatic cue-decoding processes such that those in the left hemisphere process linguistic information irrespective of acoustic cues whereas those in the right hemisphere process prosodic information.