RESUMEN
Chemical carcinogenesis is a multifactorial process comprising two main stages: initiation and promotion. Tumor promoters cause the development of tumors in initiated cells and the majority of them are non-genotoxic carcinogens. The identification of tumor promoters is important for preventing cancer. We previously identified 22 specific gene markers using a global gene expression analysis of chemically induced tumor promotion and established an in vitro real-time PCR screening assay for the assessment of the tumor promoting potential of chemicals in BALB/c 3T3 cells. Our in vitro tumor promoter screening test, based on these marker genes, enables earlier assessment, and is easier to conduct than classical methods. The general applicability of these markers, however, was unknown. In this study, to evaluate the performance of a set of markers, we independently validated a separate sample set, which had various structures and properties. Independent validation of the signature of 63 test chemicals showed an accuracy, sensitivity, and specificity of the assay of 96.8%, 97.0% and 96.7%, respectively. These results indicate that the tumor promoting activity assay, based on the expression of 22 marker genes, will become a valuable tool for rapid screening of potential tumor promoters.
Asunto(s)
Carcinógenos/toxicidad , Evaluación Preclínica de Medicamentos/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células 3T3 , Animales , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Marcadores Genéticos , Ratones , Ratones Endogámicos BALB C , Mutágenos/toxicidad , Proyectos Piloto , ARN/biosíntesis , ARN/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf-SRP54) and the N-terminal G-domain and C-terminal M-domain (pf-SRP54M) of pf-SRP54 with an amino-terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non-conserved 213-nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107-nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine-tagged pf-SRP54M bound specifically to the conserved 107-nucleotide RNA in the absence of pf-SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.
Asunto(s)
Proteínas Arqueales/genética , Proteínas de Escherichia coli/genética , Pyrococcus furiosus/genética , Proteínas de Saccharomyces cerevisiae , Partícula de Reconocimiento de Señal/genética , Secuencia de Aminoácidos , Animales , Proteínas Arqueales/metabolismo , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Proteínas de Escherichia coli/metabolismo , Humanos , Mamíferos , Metionina , Datos de Secuencia Molecular , ARN/metabolismo , Homología de Secuencia de Aminoácido , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
BACKGROUND: Previous studies have demonstrated that synthetic cell-permeable analogues of ceramide promote differentiation and inhibit proliferation of keratinocytes, and that the vitamin D3 inducible sphingomyelin cycle generates ceramide in keratinocytes. Although it has been suggested that exogenous ceramide induces apoptosis of keratinocytes, which is similar to their effect on other cell types, such as leukaemia cells, only a few studies have reported ceramide-induced apoptosis of keratinocytes. OBJECTIVE: To determine whether ceramide induces apoptosis of keratinocytes, we used the synthetic ceramide analogue, C2-ceramide (N-acetylsphingosine) and a human squamous cell carcinoma cell line, HSC-I. METHODS: We treated HSC-I cells with C2-ceramide, followed by a viability assay, morphological observations, nick end-labelling (TUNEL), DNA electrophoresis, and electron microscopy. RESULTS: In the viability assay, C2-ceramide was toxic to HSC-I cells in a dose-dependent manner. Manifestations of apoptotic morphology occurred in the ceramide-treated cells, whereas these morphological changes did not occur in cells treated with dihydroceramide (N-acetylsphinganine). TUNEL revealed that many of the ceramide-treated cells showed positive reactivity. DNA electrophoresis demonstrated that C2-ceramide caused internucleosomal fragmentation in a dose- and time-dependent manner. Electron microscopy revealed that the ceramide-treated cells manifested morphological characteristics typical of apoptosis. CONCLUSIONS: The present results demonstrate that C2-ceramide induces apoptosis of transformed human keratinocytes, whereas C2-dihydroceramide does not have such an effect. The fact that ceramide induces apoptosis of keratinocyctes raises the possibility that intracellular ceramide, which is increased with differentiation of the epidermis, might be involved in terminal differentiation, a specialized form of apoptosis of keratinocytes.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/patología , Inhibidores Enzimáticos/uso terapéutico , Neoplasias Cutáneas/patología , Esfingosina/análogos & derivados , Esfingosina/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Tamaño de la Célula , Supervivencia Celular , Ceramidas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Etiquetado Corte-Fin in Situ , Microscopía Electrónica , Neoplasias Cutáneas/tratamiento farmacológico , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Heme oxygenase is a key enzyme in the oxygen-dependent heme catabolism pathway. In order to clarify the role of highly conserved His132 in heme oxygenase isoform-1, we have prepared 30 kDa truncated rat heme oxygenase mutants in which His132 has been replaced by Ala, Gly, and Ser. The expressed recombinant mutant proteins were isolated in inclusion bodies and were recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 gel filtration column chromatography, as it eluted out of the column at the void volume. The gel filtration-purified heme oxygenase mutants have spectroscopic and enzymatic properties identical to those of wild type. The hemin complex of the H132A mutant exhibits a transition between a high-spin acid form and a low-spin alkaline form with a pKa value of 7.6 identical to that in the wild-type complex. These results demonstrate that His132 in heme oxygenase does not link to the coordinated water molecule and is not an essential residue for the enzyme activity. These results are in accordance with our previous preliminary results [Ito-Maki, M., Ishikawa, K., Mansfield Matera, K., Sato, M., Ikeda-Saito, M., & Yoshida, T. (1995) Arch. Biochem. Biophys. 317, 253-258] but contradict a recent report that His132 is the distal base linked to the coordinated water molecule and an important residue for enzyme catalysis [Wilks, A., Ortiz de Montellano, P. R., Sun, J., & Loehr, T. M. (1996) Biochemistry 35, 930-936]. Prolonged storage of the solubilized fraction from the inclusion bodies of the mutants, H132S in particular, results in an increase in the void volume fraction with a concomitant decrease of the 30 kDa fraction. We infer that His132 plays a structural role in stabilization of the heme oxygenase protein.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Histidina/metabolismo , Isoenzimas/metabolismo , Animales , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Hemo Oxigenasa (Desciclizante)/genética , Mutación , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometría Atómica , Agua/metabolismoRESUMEN
We have reported that an upstream stimulatory factor (USF) binding site is functional in transcription of the heme oxygenase-1 gene. In this study, we examined the role of USF in the induced state. By transient expression analyses with the chloramphenicol acetyl-transferase gene, we found that the USF binding site plays an important role in the induction of rat heme oxygenase-1 by cadmium, but not by hemin. To elucidate the role of USF, we prepared USF-rich nuclear extracts from control and cadmium-treated rat liver. On electrophoretic mobility shift assay using control nuclear proteins, one slowly migrating band was detected, whereas using nuclear proteins of cadmium-treated rat liver, two fast migrating bands were detected. The molecular masses of the two subunits of USF prepared from cadmium-treated rat liver were approximately 34 kDa as determined by UV cross-linking and subsequent SDS-PAGE, while the two subunits of native USF were 43 kDa and 44 kDa. DNase I footprinting analysis revealed that both the nuclear proteins bound to the same region including the USF binding site. We therefore suppose that cadmium causes some structural changes in the two proteins of USF and that the altered USF participates in the effective initiation of transcription of the rat heme oxygenase-1 gene.
Asunto(s)
Cadmio/farmacología , Proteínas de Unión al ADN , Hemo Oxigenasa (Desciclizante)/genética , Isoenzimas/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Núcleo Celular/química , Núcleo Celular/efectos de los fármacos , Reactivos de Enlaces Cruzados , Huella de ADN , Inducción Enzimática , Hemo Oxigenasa (Desciclizante)/biosíntesis , Isoenzimas/biosíntesis , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica , Ratas , Factores de Transcripción/química , Células Tumorales Cultivadas , Rayos Ultravioleta , Factores Estimuladores hacia 5'RESUMEN
A strain identified as Corynebacterium fascians was found to accumulate extracellular D-alanine from glycerol. Cultural conditions for the accumulation of D-alanine were investigated and, as a result, a yield of 7 g of D-alanine per liter was obtained after a 96-h incubation in a medium containing 5% glycerol, 4% (NH(4))(2)HPO(4), and 0.3% corn steep liquor. Optical purity of D-alanine was dependent upon the concentration of corn steep liquor. At the optimal condition, almost optically pure D-alanine was formed and readily isolated (5 g/liter) from the fermentation broth. The product was not contaminated with any detectable amount of other amino acids, except for glycine which was present at a concentration of less than 1 percent.