RESUMEN
Monocyte chemoattracant-1 (MCP-1) stimulates leukocyte chemotaxis to inflammatory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-protein-coupled seven-transmembrane receptor superfamily. These studies identified a family of antagonists, spiropiperidines. One of the more potent compounds blocks MCP-1 binding to CCR2 with a K(d) of 60 nm, but it is unable to block binding to CXCR1, CCR1, or CCR3. These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis. The compounds are effective blockers of MCP-1-driven inhibition of adenylate cyclase and MCP-1- and MCP-3-driven cytosolic calcium influx; the compounds are not agonists for these pathways. We showed that glutamate 291 (Glu(291)) of CCR2 is a critical residue for high affinity binding and that this residue contributes little to MCP-1 binding to CCR2. The basic nitrogen present in the spiropiperidine compounds may be the interaction partner for Glu(291), because the basicity of this nitrogen was essential for affinity; furthermore, a different class of antagonists, a class that does not have a basic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu(291). In addition to the CCR2 receptor, spiropiperidine compounds have affinity for several biogenic amine receptors. Receptor models indicate that the acidic residue, Glu(291), from transmembrane-7 of CCR2 is in a position similar to the acidic residue contributed from transmembrane-3 of biogenic amine receptors, which may account for the shared affinity of spiropiperidines for these two receptor classes. The models suggest that the acid-base pair, Glu(291) to piperidine nitrogen, anchors the spiropiperidine compound within the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling; thus the small molecule ligands act as antagonists. An acidic residue in transmembrane region 7 is found in most chemokine receptors and is rare in other serpentine receptors. The model of the binding site may suggest ways to make new small molecule chemokine receptor antagonists, and it may rationalize the design of more potent and selective antagonists.
Asunto(s)
Citocinas , Receptores de Citocinas/antagonistas & inhibidores , Receptores de Citocinas/química , Inhibidores de Adenilato Ciclasa , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Calcio/metabolismo , Línea Celular , Quimiocina CCL5/antagonistas & inhibidores , Quimiocina CCL7 , Quimiotaxis , Cricetinae , AMP Cíclico/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Glutámico/química , Concentración 50 Inhibidora , Cinética , Ligandos , Luciferasas/metabolismo , Datos de Secuencia Molecular , Proteínas Quimioatrayentes de Monocitos/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , Nitrógeno/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Receptores CCR2 , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Citocinas/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Transfección , Células Tumorales CultivadasRESUMEN
The CC chemokine, monocyte chemotactic protein, 1 (MCP-1) functions as a major chemoattractant for T-cells and monocytes by interacting with the seven-transmembrane G protein-coupled receptor CCR2. To identify which residues of MCP-1 contribute to signaling though CCR2, we mutated all the surface-exposed residues to alanine and other amino acids and made some selective large changes at the amino terminus. We then characterized the impact of these mutations on three postreceptor pathways involving inhibition of cAMP synthesis, stimulation of cytosolic calcium influx, and chemotaxis. The results highlight several important features of the signaling process and the correlation between binding and signaling: The amino terminus of MCP-1 is essential as truncation of residues 2-8 ([1+9-76]hMCP-1) results in a protein that cannot stimulate chemotaxis. However, the exact peptide sequence may be unimportant as individual alanine mutations or simultaneous replacement of residues 3-6 with alanine had little effect. Y13 is also important and must be a large nonpolar residue for chemotaxis to occur. Interestingly, both Y13 and [1+9-76]hMCP-1 are high-affinity binders and thus affinity of these mutants is not correlated with ability to promote chemotaxis. For the other surface residues there is a strong correlation between binding affinity and agonist potency in all three signaling pathways. Perhaps the most interesting observation is that although Y13A and [1+9-76]hMCP are antagonists of chemotaxis, they are agonists of pathways involving inhibition of cAMP synthesis and, in the case of Y13A, calcium influx. These results demonstrate that these two well-known signaling events are not sufficient to drive chemotaxis. Furthermore, it suggests that specific molecular features of MCP-1 induce different conformations in CCR2 that are coupled to separate postreceptor pathways. Therefore, by judicious design of antagonists, it should be possible to trap CCR2 in conformational states that are unable to stimulate all of the pathways required for chemotaxis.
Asunto(s)
Aminoácidos/fisiología , Quimiocina CCL2/fisiología , Receptores de Quimiocina/fisiología , Receptores de Citocinas/fisiología , Transducción de Señal , Aminoácidos/aislamiento & purificación , Sitios de Unión/genética , Calcio/antagonistas & inhibidores , Calcio/metabolismo , Línea Celular , Membrana Celular/genética , Membrana Celular/fisiología , Inhibición de Migración Celular , Quimiocina CCL2/agonistas , Quimiocina CCL2/genética , AMP Cíclico/antagonistas & inhibidores , Humanos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Receptores CCR2 , Receptores de Quimiocina/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal/genética , Tirosina/genética , Tirosina/fisiologíaRESUMEN
The CC chemokine, MCP-1, has been identified as a major chemoattractant for T cells and monocytes, and plays a significant role in the pathology of inflammatory diseases. To identify the regions of MCP-1 that contact its receptor, CCR2, we substituted all surface-exposed residues with alanine. Some residues were also mutated to other amino acids to identify the importance of charge, hydrophobicity, or aromaticity at specific positions. The binding affinity of each mutant for CCR2 was assayed with THP-1 and CCR2-transfected CHL cells. The majority of point mutations had no effect. Residues at the N-terminus of the protein, known to be crucial for signaling, contribute less than a factor of 10 to the binding affinity. However, two clusters of primarily basic residues (R24, K35, K38, K49, and Y13), separated by a 35 A hydrophobic groove, reduced the level of binding by 15-100-fold. A peptide fragment encompassing residues 13-35 recapitulated some of the mutational data derived from the intact protein. It exhibited modest binding as a linear peptide and dramatically improved affinity when the region which adopts a single turn of a 3(10)-helix in the protein, which includes R24, was constrained by a disulfide bond. Additional constraints at the ends of the peptide, corresponding to the disulfide between the first and third cysteines in MCP-1, yielded further improvements in affinity. Together, these data suggest a model in which a large surface area of MCP-1 contacts the receptor, and the accumulation of a number of weak interactions results in the 35 pM affinity observed for the wild-type (WT) protein. The receptor binding site of MCP-1 also is significantly different from the binding sites of RANTES and IL-8, providing insight into the issue of receptor specificity. It was previously shown that the N-terminus of CCR2 is critical for binding MCP-1 [Monteclaro, F. S., and Charo, I. F. (1996) J. Biol. Chem. 271, 19084-92; Monteclaro, F. S., and Charo, I. F. (1997) J. Biol. Chem. 272, 23186-90]. Point mutations of six acidic residues in this region of the receptor were made to test their role in ligand binding. This identified D25 and D27 of the DYDY motif as being important. On the basis of our data, we propose a model in which the receptor N-terminus lies along the hydrophobic groove in an extended fashion, placing the DYDY motif near the basic cluster involving R24 and K49 of MCP-1. This in turn orients the signaling residues (Y13 and the N-terminus) for productive interaction with the receptor.
Asunto(s)
Quimiocina CCL2/química , Quimiocina CCL2/metabolismo , Receptores de Quimiocina/metabolismo , Receptores de Citocinas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Quimiocina CCL2/genética , Cricetinae , Cricetulus , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptores CCR2 , Receptores de Quimiocina/química , Receptores de Citocinas/químicaRESUMEN
To address the role of dimerization in the function of the monocyte chemoattractant protein-1, MCP-1, we mutated residues that comprise the core of the dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b. One mutant, P8A*, does not dimerize. However, it has wild type binding affinity, stimulates chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx with wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx, and is unable to stimulate chemotaxis. Thus Tyr13 may make important contacts with the receptor that are required for high affinity binding and signal transduction. We also explored whether a mutant, [1+9-76]MCP-1 (MCP-1 lacking residues 2-8), antagonizes wild type MCP-1 by competitive inhibition, or by a dominant negative mechanism wherein heterodimers of MCP-1 and [1+9-76]MCP-1 bind to the receptor but are signaling incompetent. Consistent with the finding that MCP-1 can bind and activate the receptor as a monomer, we demonstrate that binding of MCP-1 in the presence of [1+9-76]MCP-1 over a range of concentrations of both ligands fits well to a simple model in which monomeric [1+9-76]MCP-1 functions as a competitive inhibitor of monomeric MCP-1. These results are crucial for elucidating the molecular details of receptor binding and activation, for interpreting mutagenesis data, for understanding how antagonistic chemokine variants function, and for the design of receptor antagonists.
Asunto(s)
Quimiocina CCL2/metabolismo , Receptores de Quimiocina , Receptores de Citocinas/metabolismo , Dimerización , Disulfuros/química , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Receptores CCR2 , Receptores de Citocinas/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
In an attempt to verify the relative efficacy of early concurrent vs. sequential timing of thoracic radiotherapy (TRT) and platinum/etoposide chemotherapy, 48 patients with limited-stage small cell lung cancer treated with either early-concurrent (29 patients) or sequential (19 patients) TRT and platinum/etoposide chemotherapy were evaluated. Disease-specific prognostic variables and the role of prophylactic cranial irradiation (PCI) were also analyzed. Thirty-four patients (71%) received TRT to a dose of 45 Gy in 25 fractions (range, 30-55 Gy). Most patients (75%) received 4-6 cycles of chemotherapy. Twenty-one of 27 patients achieving a complete response after completion of TRT and chemotherapy received PCI. Median follow-up was 29.3 months (range, 12-98 months). Variables of potential prognostic significance were evaluated by both univariate and multivariate analysis. The absolute and relapse-free survival rates for all patients were 42% and 35% at 2 years and 32% and 31% at 5 years, respectively. Thirty-six sites of failure were observed in 27 patients. Thoracic recurrence occurred in nine patients, and the central nervous system (CNS) was the most common site of distant failure (15 patients). Multivariate analysis demonstrated that (a) early concurrent TRT and chemotherapy vs. chemotherapy followed by sequential TRT and (b) disease volume [less than or greater than one-third of the thoracic width] were significantly predictive for survival (P=0.036 and P=0.05, respectively). Rates of control of thoracic disease were 79% for patients with a disease volume less than one-third of the thoracic width vs. 36% for disease volumes greater than one-third of the thoracic width (P=0.0009). Early concurrent TRT and chemotherapy resulted in a significantly lower incidence of distant metastasis (26% for concurrent vs. 63% for sequential; P=0.008). In patients who received PCI, the CNS control rate was 86% vs. 56% in patients not treated with PCI. Our findings suggest that (a) treatment with early concurrent TRT and platinum/etoposide chemotherapy may improve survival when compared with sequential treatment and (b) PCI for patients with complete systemic responses is effective in preventing CNS recurrence. We also conclude that thoracic disease volume is a significant prognostic factor for both local control and overall survival.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/radioterapia , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/radioterapia , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/radioterapia , Anciano , Antineoplásicos/administración & dosificación , Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias Encefálicas/secundario , Carcinoma de Células Pequeñas/secundario , Terapia Combinada , Supervivencia sin Enfermedad , Esquema de Medicación , Etopósido/administración & dosificación , Femenino , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Compuestos de Platino/administración & dosificación , Valor Predictivo de las Pruebas , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
OBJECTIVES: To determine the long-term results of a bladder-sparing approach in the treatment of muscle-invasive bladder cancer. METHODS: Ninety-four patients with invasive transitional cell carcinoma of the bladder were treated by transurethral resection followed by 2 or 3 cycles of cisplatin-based chemotherapy. Patients were then treated with 6480 cGy of radiation in 49 patients, segmental cystectomy in 8, or surveillance only in 7. Patients who failed to respond to chemotherapy or radiation therapy, or who developed recurrent muscle-invasive disease in follow-up, underwent salvage cystectomy. Patients were then carefully followed for a median follow-up of more than 5 years. RESULTS: After initial therapy, 53 patients (56%) were alive with their bladder preserved. Thirty of those 53 (57%) developed a local recurrence in follow-up. After a median follow-up of more than 5 years, the ultimate relapse-free survival is 49% (Stage T2, 84%; T3, 53%; and T4, 11%; P < 0.01). Of all patients enrolled, 53% had bladder preservation; however, of the currently surviving patients, 16 of 39 (41%) have their bladders intact (T2, 50%; T3, 37%; T4, 0%). Only 18% of the initially enrolled population is alive with a preserved bladder. The 5-year survival of patients who had cystectomy at some point during the study, compared with those who have had their bladders preserved, was 65% versus 40%, respectively (P < 0.01). CONCLUSIONS: Our long-term results with multimodality therapy with attempted bladder preservation showed no definitive improvement in survival compared with modern series of cystectomy alone, and a disappointingly low rate of disease-free bladder preservation. We found a high rate of locally recurrent disease in the preserved bladders. We also noted a decrease in survival in our patients with bladder preservation compared with those without preservation. Bladder preservation, although possible, should be limited to a very select group of patients.
Asunto(s)
Carcinoma de Células Transicionales/cirugía , Neoplasias de la Vejiga Urinaria/cirugía , Anciano , Anciano de 80 o más Años , Carcinoma de Células Transicionales/mortalidad , Carcinoma de Células Transicionales/patología , Carcinoma de Células Transicionales/terapia , Terapia Combinada , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Músculo Liso , Invasividad Neoplásica , Análisis de Supervivencia , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/terapiaRESUMEN
c-Src is a nontransforming tyrosine kinase that participates in signaling events mediated by a variety of polypeptide growth factor receptors, including the epidermal growth factor receptor (EGFR). Overexpression and continual ligand stimulation of the EGFR results in morphological transformation of cells in vitro and tumor development in vivo. Elevated levels of c-Src and the EGFR are found in a variety of human malignancies, raising the question of whether c-Src can functionally cooperate with the EGFR during tumorigenesis. To address this issue, we generated c-Src/EGFR double overexpressors and compared their proliferative and biochemical characteristics to those of single overexpressors and control cells. We found that in cells expressing high levels of receptor, c-Src potentiated DNA synthesis, growth in soft agar, and tumor formation in nude mice. Growth potentiation was associated with the formation of a heterocomplex between c-Src and activated EGFR, the appearance of a distinct tyrosyl phosphorylation on the receptor, and an enhancement of receptor substrate phosphorylation. These findings indicate that c-Src is capable of potentiating receptor-mediated tumorigenesis and suggest that synergism between c-Src and the EGFR may contribute to a more aggressive phenotype in multiple human tumors.
Asunto(s)
Transformación Celular Neoplásica , Receptores ErbB/metabolismo , Neoplasias/etiología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Animales , Biomarcadores de Tumor , Neoplasias de la Mama/etiología , Carcinoma/etiología , Células Cultivadas , Neoplasias del Colon/etiología , Sinergismo Farmacológico , Ratones , Mitosis , Fosforilación , Unión Proteica , Proteínas Tirosina Quinasas/farmacología , Proteínas Proto-Oncogénicas pp60(c-src)/farmacologíaRESUMEN
Leflunomide is a novel and effective immunosuppressant that holds promise as a therapeutic agent, but the mechanism of action is unknown. Here we provide evidence that leflunomide is a general cytostatic agent for a wide range of cells. The IC50 for proliferation in transformed cell lines ranged from 2 to 16 microM. The mean IC50 for proliferation of mitogen-stimulated rat lymphocytes (86 nM) was much lower than for mouse (3.5 microM) or human (12.5 microM) lymphocytes. Initial signal transduction events (epidermal growth factor receptor-stimulated phosphotyrosine formation and phytohemagglutinin-stimulated Ca++ mobilization) were unaffected by antiproliferative concentrations of leflunomide. Leflunomide was equally as effective against mitogenic stimuli that bypass initial signaling events as it was against surface receptor-mediated mitogens. Leflunomide was fully potent when added 8 hr after the mitogenic stimulus. Cytokine dependent T-cell growth also was blocked by leflunomide. Leflunomide caused only partial reductions of autocrine cytokine production or cytokine receptor expression. Leflunomide blocked completely the progression of rat lymphocytes beyond early S-phase of cell cycle and inhibited entry of human T-cells into the G2 and M-phases without causing cell death. Inhibition of proliferation could not be reversed by purine nucleosides. The results suggest that leflunomide's mechanism of action differs from that of other immunosuppressive agents such as corticosteroids, Cyclosporine A, rapamycin or mycophenolic acid.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Inmunosupresores/farmacología , Isoxazoles/farmacología , Activación de Linfocitos/efectos de los fármacos , Animales , Antígenos de Diferenciación de Linfocitos T/metabolismo , Calcio/metabolismo , Femenino , Técnicas In Vitro , Interleucina-2/metabolismo , Leflunamida , Ratones , Ratones Endogámicos BALB C , Fosfotirosina , Proteínas Tirosina Quinasas/metabolismo , Ratas , Receptores de Interleucina-2/metabolismo , Células Tumorales Cultivadas , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
A phase III clinical trial was developed to test whether the addition of etoposide to a high-dose cytosine arabinoside regimen would improve the remission rate, duration of remission, and survival in relapsed and refractory patients with acute myelogenous leukemia. One hundred and thirty-one patients stratified by age, performance status, percentage of marrow blasts, platelet count, bilirubin and presence or absence of clinical infection, refractory or relapsed (+/- 9 months) were randomized to receive high-dose cytosine arabinoside, 3 g/m2 every 12 h for 6 days with or without three doses of etoposide, 100 mg/m2 days 7-9. Of 67 patients randomized to cytosine arabinoside alone, 31% obtained a complete remission with a median remission duration of 11.9 months. Of 66 patients randomized to the combination regimen, 38% obtained a complete remission with a median duration of 25 months. None of these differences were statistically significant. Significantly (p = 0.036) longer survival was seen in patients on the combination regimen under the age of 50. There was no difference in overall survival. Six and 8%, respectively, of patients were free of disease at 5 years. The addition of etoposide to a high-dose cytosine arabinoside regimen had at best a marginal effect at the expense of some increase in toxicity.
Asunto(s)
Citarabina/administración & dosificación , Etopósido/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Citarabina/efectos adversos , Etopósido/efectos adversos , Humanos , Análisis de SupervivenciaRESUMEN
The treatment of myelodysplastic disorders with vitamins A and D or derivatives and nutrients has been less than satisfactory. Combinations of vitamin D3 and 13-CRA with or without cytosine arabinoside appear to offer no advantage over any of the single agents alone. Until other vitamin D derivatives are developed that are effective but do not cause hypercalcemia, vitamin D3 cannot be recommended for the treatment of MDS. 13-CRA has been shown to be effective in some patients with MDS; however, it cannot be recommended as standard therapy because of the conflicting data cited above. Further clinical trials with 13-CRA perhaps in combination with vitamin E or colony-stimulating factors are clearly indicated for this disease for which we have no effective therapy.
Asunto(s)
Síndromes Mielodisplásicos/dietoterapia , Vitamina A/uso terapéutico , Vitamina D/uso terapéutico , HumanosAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Transicionales/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Anciano , Carcinoma de Células Transicionales/mortalidad , Cisplatino/administración & dosificación , Ensayos Clínicos como Asunto , Terapia Combinada , Cistectomía , Doxorrubicina/administración & dosificación , Femenino , Humanos , Masculino , Metotrexato/administración & dosificación , Radioterapia de Alta Energía , Neoplasias de la Vejiga Urinaria/mortalidad , Vinblastina/administración & dosificaciónRESUMEN
Lipid profiles were determined in 56 elderly patients with benign essential hypertension during an open-label 1-year study of the safety and efficacy of isradipine, a new calcium antagonist, in controlling blood pressure. Patients with diastolic blood pressures between 96 and 115 mm Hg were titrated with isradipine (mean dose of 11 mg/day) to reduce blood pressure to less than 90 mm Hg. Ten of these patients received concomitant hydrochlorothiazide (HCTZ) 50 mg/day for additional control. Sera were analyzed using standard methods at the end of a 2- to 4-week washout period, and at the end of Months 6 and 12, for total cholesterol (CHOL) and HDL- and LDL-cholesterol. Changes in lipid values (mg/dl) from baseline to 12 months with isradipine alone (n = 38) were as follows: CHOL, -7.5; HDL, +3.9 (p less than 0.05); LDL, -6.2; CHOL/HDL, -0.6 (p less than 0.05). For patients receiving concomitant HCTZ (n = 9), the changes were as follows: CHOL, -4.9; HDL, +3.4; LDL, -16.8; CHOL/HDL, -0.4. In conclusion, isradipine alone was associated with significant improvements in HDL cholesterol and total CHOL/HDL ratio. Lipid profiles of patients receiving isradipine and HCTZ were minimally affected. Favorable lipid changes with isradipine suggest that it may have advantages in the treatment of hypertensive patients.
Asunto(s)
Hidroclorotiazida/uso terapéutico , Hipertensión/sangre , Lípidos/sangre , Piridinas/uso terapéutico , Anciano , Anciano de 80 o más Años , Presión Sanguínea/efectos de los fármacos , Colesterol/sangre , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Quimioterapia Combinada , Femenino , Humanos , Hidroclorotiazida/efectos adversos , Hipertensión/tratamiento farmacológico , Isradipino , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Piridinas/efectos adversosRESUMEN
Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds and transforms the corresponding cells in culture. Neoplastic transformation by AEV is mediated principally by an oncogene known as v-erb-B. We have explored the means by which this gene is expressed from the genome of AEV and uncovered an important structural determinant for the potency of the oncogene. In order to define the boundaries of v-erb-B and the supplementary oncogene, v-erb-A, we sequenced all but a small portion of the genome of the ES4 strain of AEV. We then demonstrated that, during expression in infected cells, splicing fuses the first six amino acids of the retroviral gene gag to the body of the v-erb-B protein. In order to explore the impact of this fusion on the function of v-erb-B, we constructed vectors with Murine Leukemia Virus that express the oncogene either with or without the fusion to gag. Viruses generated from these two vectors differed greatly in their abilities to transform cells: fusion of v-erb-B with gag enhanced its transforming ability 50 to 100-fold as determined by focus transformation assays and growth in soft agar. Our data suggest that the difference in transforming ability is not due to alterations in transcription or translation but, rather, may result from changes in post-translational modification.
Asunto(s)
Transformación Celular Neoplásica , Genes gag , Oncogenes , Proteínas Oncogénicas de Retroviridae/genética , Retroviridae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , Proteínas Oncogénicas v-erbB , Proteínas Oncogénicas Virales , Ratas , Proteínas Virales de Fusión/fisiologíaRESUMEN
Two patients presenting with anasarca were found to have aggressive B-cell lymphoma. No bulky disease was detected. The diagnosis was rapidly established by the flow cytometric analysis of cell surface immunophenotype and cell cycle fractions of pleural or peritoneal cells. Such presentation of lymphoma is unusual and previously undescribed, and it may have a significant negative prognostic impact. The authors' observations indicate that lymphoma be included in the differential diagnosis of anasarca and that flow cytometry can be useful for a fast confirmation of the diagnosis.
Asunto(s)
Edema/etiología , Linfoma/diagnóstico , Anciano , Antígenos CD/análisis , Linfocitos B , ADN de Neoplasias/análisis , Citometría de Flujo , Humanos , Linfoma/complicaciones , Linfoma/inmunología , Masculino , Persona de Mediana EdadRESUMEN
Remarkable advances in the treatment of urologic malignancies have recently been made. Monoclonal antibodies selective for a variety of normal and malignant urologic tissues have been useful in defining normal antigens and tumor-associated antigens and have potential as diagnostic and immunotherapeutic agents. In renal cancer, monoclonal antibodies can define serum markers, radiolabel tumor xenografts, and assist in specific tissue diagnosis. Additionally, there is potential for these antibodies either alone or as conjugates to localize and kill tumors. Monoclonal antibodies to bladder cancer associated antigens are able to demonstrate differential antigen expression on superficial versus invasive tumors, to refine urinary cytologic diagnosis of bladder cancer, and to predict invasive recurrence of superficial cancer. Monoclonal antibodies have localized bladder tumor xenografts and can inhibit tumor growth when conjugated to radioisotopes or toxins. In prostate cancer monoclonal antibodies to prostate antigens are not usually tumor specific. Monoclonal antibodies to prostate antigen (PA) and prostatic acid phosphatase (PAP) are able to localize prostate cancer metastases. Chemotherapy-conjugated anti-PAP monoclonal antibodies have demonstrable inhibition on human prostate cancer xenografted tumor growth. Monoclonal antibodies have defined normal and tumor-associated antigens in urologic cancers and are expected to be useful in immunodiagnosis and cancer therapy in the near future.
Asunto(s)
Anticuerpos Monoclonales , Neoplasias Urológicas/diagnóstico , Fosfatasa Ácida/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Humanos , Neoplasias Urológicas/tratamiento farmacológicoRESUMEN
Arteriovenous malformations are rare, but, considering the life-threatening consequences of these lesions, they must constantly be considered in a differential diagnosis. The management of this patient from the initial symptoms emphasizes the need for a methodical approach to oral diagnosis using the fundamental techniques of inspection, auscultation, and palpation, as well as the more recent techniques of laboratory and radiographic evaluation.
Asunto(s)
Malformaciones Arteriovenosas/diagnóstico por imagen , Mandíbula/irrigación sanguínea , Adulto , Diagnóstico Diferencial , Femenino , Humanos , RadiografíaRESUMEN
Initial treatment of elderly hypertensive patients with an angiotensin-converting enzyme inhibitor is currently discouraged due to such patients' typical low-renin profile. To validate this principle, we studied 38 elderly males (aged greater than or equal to 65 years) with mild to moderate hypertension, comparing hemodynamic responses to and subjective impressions of enalapril or hydrochlorothiazide (HCTZ). After gradual withdrawal of existing antihypertensive therapy and a four-week, single-blind placebo period, each patient was randomized in a double-blind fashion to receive either enalapril 10-20 mg/d or HCTZ 12.5-25 mg/d for two to four weeks. Combination therapy with both agents was employed if either alone failed to reduce seated diastolic BP to less than or equal to 90 mm Hg. Equivalent proportions of patients receiving enalapril or HCTZ (8 of 19 and 10 of 19, respectively; p = ns) responded with significant reductions in systolic and diastolic BP in seated and standing positions. Combination therapy was most effective in patients receiving HCTZ prior to enalapril. In patients receiving enalapril before HCTZ, BP changes were minimal. No adverse effects were observed in the enalapril group but occurred in an equivalent fraction of patients in the other groups (4 of 10 HCTZ alone, 6 of 20 enalapril + HCTZ; p = ns). We conclude that enalapril may be considered a reasonable monotherapeutic antihypertensive agent in some elderly patients. Combination with HCTZ is beneficial in patients who fail to respond adequately to HCTZ alone.
Asunto(s)
Enalapril/uso terapéutico , Hidroclorotiazida/uso terapéutico , Hipertensión/tratamiento farmacológico , Anciano , Ensayos Clínicos como Asunto , Método Doble Ciego , Enalapril/efectos adversos , Humanos , Hidroclorotiazida/efectos adversos , Masculino , Distribución AleatoriaRESUMEN
Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that causes erythroblastosis and sarcomas in chickens and transforms immature erythroid cells and fibroblasts in culture. AEV encodes two oncogenes, v-erbA and v-erbB, whose products are closely related to the thyroxine receptor and the epidermal growth factor receptor, respectively. Since tyrosine protein kinases have been implicated in the process of normal growth signal transduction, we wished to study the possible consequences of the expression of these mutated, growth-regulating receptor genes on the activity of the cellular tyrosine kinase pp60c-src. A continuous cell line from AEV-infected quail embryo fibroblasts was derived that exhibited a typical transformed phenotype and expressed the viral oncogene products, p75gag-erbA and gp66-68erbB. Using an immune-complex kinase assay, we found that the specific activity of pp60c-src in AEV-transformed quail cells was decreased by a factor of 6-30 relative to that found in uninfected quail cells. A concomitant 50-80% reduction of 32Pi incorporation into the pp60c-src protein from radiolabeled, transformed cells was also observed, indicating a relationship between hypophosphorylation and diminished enzyme activity. Partial proteolytic phosphopeptide analysis revealed a decrease in phosphorylation of both serine- and tyrosine-containing peptides, suggesting an activation of specific phosphatases or inhibition of specific kinases in the AEV-transformed quail cells. Similar results were found in pp60c-src precipitated from AEV-transformed chicken and rat cells.
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Leucosis Aviar/enzimología , Proteínas Tirosina Quinasas/metabolismo , Proteínas de los Retroviridae/metabolismo , Alpharetrovirus , Animales , Pollos , Coturnix , Regulación de la Expresión Génica , Proteína Oncogénica pp60(v-src) , Oncogenes , Mapeo Peptídico , Fosforilación , Ratas , Serina/análisis , Tirosina/análisisRESUMEN
A monoclonal antibody, R2D2, raised to the src gene product of Rous sarcoma virus was found to inhibit the tyrosine protein kinase activity of pp60v-src in autophosphorylation reactions and in reactions involving exogenously added substrates, such as casein and histone. R2D2 also inhibited the enzymatic activity of two related viral transforming proteins, pp70gag-fgr and pp90gag-yes. The inhibitory ability of R2D2 was dependent upon immunoglobulin concentration and could be demonstrated in both immune complexes formed directly with R2D2 and preformed immune complexes to which R2D2 was added. Binding sites in both the amino-terminal 110 amino acid residues and the carboxy-terminal 240 amino acids of pp60v-src were identified for R2D2. These results indicate that at least part of the epitope recognized by R2D2 resides within a region of the src protein which is required for protein kinase activity. The localization of the R2D2 epitope to the amino- as well as to the carboxy-terminal portions of pp60v-src, together with results of studies analyzing the relative binding efficiencies of R2D2 to the intact protein and to V-8 proteolytic fragments of pp60v-src, are consistent with the view that the R2D2 epitope is conformational in nature and that it is assembled from residues contained within both N-terminal and C-terminal regions of the molecule.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Oncogénicas de Retroviridae , Proteínas de los Retroviridae/antagonistas & inhibidores , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Reacciones Cruzadas , Epítopos/inmunología , Proteína Oncogénica pp60(v-src) , Fragmentos de Péptidos/inmunología , Fosforilación , Unión Proteica , Proteínas Tirosina Quinasas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas de los Retroviridae/inmunología , Homología de Secuencia de Ácido NucleicoRESUMEN
A 60-year-old man with metastatic transitional cell carcinoma of the bladder was treated with weekly methotrexate injections, each followed by leucovorin rescue. After two to three hours of sun exposure, he experienced a mild sunburn erythema on his lower extremities in areas not chronically sun exposed. He received methotrexate the next day. More than one week later, following another dose of methotrexate, he experienced a second-degree burn in the areas of the prior sunburn, with sparing of chronically exposed areas on his face, neck, and arms. We discuss here the reactivation and exacerbation of sunburn by methotrexate and other chemotherapeutic agents, the unique features of delayed onset and selective skin involvement in our case, and the need for sun protection in patients receiving these drugs.