RESUMEN
Mercury has harmful effects in both rodents and humans. In rodent tissue culture cells exposed to HgCl(2), the metal ions were observed to concentrate in cell nuclei and to associate with chromatin. Thus, transcription factors and other proteins associated with chromatin are possible targets of mercuric ion toxicity. In this study, mercuric ions were found to inhibit the DNA binding activity of the Cys(2)His(2) zinc finger proteins transcription factor IIIA (TFIIIA) and Sp1. These factors are prototypes of the largest eukaryotic protein superfamily. Neither the presence of excess zinc ions nor beta-mercaptoethanol prevented inhibition by mercuric ions. Mercuric ions also inhibited DNA binding by the non-zinc finger protein AP2. Zinc finger-DNA binding was inhibited when both TFIIIA/5S RNA complex and TFIIIA alone were preincubated with concentrations as low as 15 microM mercuric ion. Inhibition occurred in less than 1 min and was not readily reversible. Mercuric ions also inhibited the digestion of DNA by the restriction enzymes BamHI or EcoRI. Inhibition of transcription factors as well as potentially other DNA binding proteins by micromolar concentrations of mercuric ion suggests additional biochemical mechanisms for mercury toxicity in promoting disease via alterations in gene transcription patterns.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/efectos de los fármacos , Mercurio/farmacología , Factores de Transcripción/metabolismo , Animales , ADN/metabolismo , Enzimas de Restricción del ADN/metabolismo , Proteínas de Unión al ADN/química , Interacciones Farmacológicas , Femenino , Ovario/efectos de los fármacos , Ovario/fisiología , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción AP-2 , Factor de Transcripción TFIIIA , Factores de Transcripción/química , Proteínas de Xenopus , Xenopus laevis , Zinc/farmacología , Dedos de Zinc/efectos de los fármacosRESUMEN
The combination of weekly irinotecan (CPT-11) and monthly cisplatin has shown promising activity in advanced non-small cell lung cancer (NSCLC) in previous Phase I and II studies. However, same-day administration of these agents may better exploit their therapeutic synergy and minimize toxicities. This multicenter Phase II study was undertaken to evaluate the efficacy and safety of a combination of weekly CPT-11 and weekly cisplatin in patients with advanced NSCLC. Patients with chemotherapy-naive stage IIIB or IV NSCLC were treated with repeated cycles of therapy comprising weekly treatment with both cisplatin and CPT-11 for 4 weeks, followed by a 2-week rest. The starting doses of CPT-11 and cisplatin were 65 and 30 mg/m2, respectively. Treatment was continued until the occurrence of disease progression, unacceptable toxicity, or a maximum of six cycles. Fifty patients were enrolled. The median age was 59 years (range, 44-79 years). Eastern Cooperative Oncology Group performance status was 0 in 22 patients, 1 in 19 patients, and 2 in 9 patients. Seven and 43 patients had stages IIIB and IV disease, respectively. Five patients had brain metastasis. Patients received a median of three 6-week cycles (range, 1-6). The objective response rate was 36% (18 of 50; 95% confidence interval, 24-54%) and included 18 partial responses. Median time to tumor progression was 6.9 months (range, 0.6-15.2). The median survival was 11.6 months (range, 0.16-21.9 months), and the 1-year survival rate was 46%. Grade 3/4 nonhematological toxicities included vomiting (12%) and diarrhea (26%). Grade 3/4 hematological toxicities included anemia (14%), neutropenia (26%), and thrombocytopenia (14%). Relative dose intensities for CPT-11 and cisplatin were 89 and 62%, respectively. Weekly combined administration of CPT-11 and cisplatin achieved a promising overall response rate, median time to tumor progression, and median survival in patients with stage IIIB/IV NSCLC. The regimen was well tolerated, and the planned dose intensity was well maintained. Further evaluation of this combination in NSCLC is warranted.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Camptotecina/análogos & derivados , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Adulto , Anciano , Camptotecina/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Cisplatino/administración & dosificación , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Irinotecán , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Transcription factor IIIA (TFIIIA) activates 5S ribosomal RNA gene transcription in eukaryotes. The protein from vertebrates has nine contiguous Cys(2)His(2)zinc fingers which function in nucleic acid binding, and a C-terminal region involved in transcription activation. In order to identify protein partners for TFIIIA, yeast two-hybrid screens were performed using the C-terminal region of Xenopus TFIIIA as an attractor and a rat cDNA library as a source of potential partners. A cDNA clone was identified which produced a protein in yeast that interacted with Xenopus TFIIIA but not with yeast TFIIIA. This rat clone was sequenced and the primary structure of the human homolog (termed TFIIIA-intP for TFIIIA-interacting protein) was determined from expressed sequence tags. In vitro interaction of recombinant human TFIIIA-intP with recombinant Xenopus TFIIIA was demonstrated by immuno-precipitation of the complex using anti-TFIIIA-intP antibody. Interaction of rat TFIIIA with rat TFIIIA-intP was indicated by co-chromatography of the two proteins on DEAE-5PW following fractionation of a rat liver extract on cation, anion and gel filtration resins. In a HeLa cell nuclear extract, recombinant TFIIIA-intP was able to stimulate TFIIIA-dependent transcription of the Xenopus 5S ribosomal RNA gene but not TFIIIA-independent transcription of the human adenovirus VA RNA gene.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Extractos Hepáticos/química , Extractos Hepáticos/metabolismo , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Unión Proteica , ARN Ribosómico 5S/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factor de Transcripción TFIIIA , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , XenopusRESUMEN
We propose a new passive optical thresholding device that combines the principles of multimode interference (MMI) with self-guiding. The multimode region is composed of a nonlinear optical material that will support a self-guided beam (i.e., a material with a positive Kerr nonlinearity). The device operates by switching between the MMI mode of operation and the self-guiding mode of operation, depending on the input light intensity. We describe the basic principles of a self-guiding MMI device, simulate the device, and discuss design issues associated with these optically controlled optical switches.
RESUMEN
The association of lead with chromatin in cells suggests that deleterious metal effects may in part be mediated through alterations in gene function. To elucidate if and how lead may alter DNA binding of cysteine-rich zinc finger proteins, lead ions were analyzed for their ability to alter the DNA binding mechanism of the Cys(2)His(2) zinc finger protein transcription factor IIIA (TFIIIA). As assayed by DNase I protection, the interaction of TFIIIA with the 50-bp internal control region of the 5S ribosomal gene was partially inhibited by 5 microM lead ions and completely inhibited by 10 to 20 microM lead ions. Preincubation of free TFIIIA with lead resulted in DNA-binding inhibition, whereas preincubation of a TFIIIA/5S RNA complex with lead did not result in DNA-binding inhibition. Because 5S RNA binds TFIIIA zinc fingers, this result is consistent with an inhibition mechanism via lead binding to zinc fingers. The complete loss of DNase I protection on the 5S gene indicates the mechanism of inhibition minimally involves the N-terminal fingers of TFIIIA. Inhibition was not readily reversible and occurred in the presence of an excess of beta-mercaptoethanol. Inhibition kinetics were fast, progressing to completion in approximately 5 min. Millimolar concentrations of sulfhydryl-specific arsenic ions were not inhibitory for TFIIIA binding. Micromolar concentrations of lead inhibited DNA binding by Sp1, another Cys(2)His(2) finger protein, but not by the nonfinger protein AP2. Inhibition of Cys(2)His(2) zinc finger transcription factors by lead ions at concentrations near those known to have deleterious physiological effects points to new molecular mechanisms for lead toxicity in promoting disease.