RESUMEN
We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in non-small cell lung cancer. Over 226,000 SAGE tags were sequence analyzed from two independent primary lung cancers and two normal human bronchial/tracheal epithelial cell cultures. A total of 226,000 SAGE tags were sequence identified, representing 43,254 unique transcripts. Comparison of the tags present in the tumor with those identified in the normal tissue revealed 175 transcript tags that were overrepresented in the normal tissue and 142 tags that were overexpressed in the tumor by 10-fold or more. Northern hybridization was performed on 15 of the most abundantly expressed tags identified in the tumors. These tags were derived from either a known gene or a matched expressed sequence tag clone. The transcripts for 3 of the 15 genes, PGP 9.5, B-myb, and human mutT, were abundantly expressed in primary lung cancers (10 of 18, 15 of 18, and 6 of 12 tumors, respectively). In contrast, the presence of PGP9.5 and B-myb was much less frequent in primary tumors derived from other tissue origins. These results suggest that at least a portion of the transcripts identified by SAGE are frequently associated with lung cancer, and that their overexpression may contribute to lung tumorigenesis. The identification and further characterization of genes generated by SAGE should provide potential new targets for the diagnosis, prognosis, and therapy of lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Escamosas/genética , Expresión Génica , Técnicas Genéticas , Neoplasias Pulmonares/genética , Bronquios , Células Cultivadas , Humanos , ARN Mensajero/análisis , Lugares Marcados de Secuencia , TráqueaRESUMEN
Serial analysis of gene expression (SAGE) allows for a quantitative, representative, and comprehensive profile of gene expression. We have utilized SAGE technology to contrast the differential gene expression profile in rat embryo fibroblast cells producing temperature-sensitive p53 tumor suppressor protein at permissive or non-permissive temperatures. Analysis of approximately 15,000 genes revealed that the expression of 14 genes (P < 0.001, > or = 0.03% abundance) was dependent on functional p53 protein, whereas the expression of three genes was significantly higher in cells producing non-functional p53 protein. Those genes whose expression was increased by functional p53 include RAS, U6 snRNA, cyclin G, EGR-1, and several novel genes. The expression of actin, tubulin, and HSP70 genes was elevated at the non-permissive temperature for p53 function. Interestingly, the expression of several genes was dependent on a non-temperature-sensitive mutant p53 suggesting altered transcription profiles dependent on specific p53 mutant proteins. These results demonstrate the utility of SAGE for rapidly and reproducibly evaluating global transcriptional responses within different cell populations.
Asunto(s)
Clonación Molecular/métodos , Regulación de la Expresión Génica , Genes p53 , Transcripción Genética , Animales , División Celular/genética , Línea Celular , Embrión de Mamíferos , Fibroblastos/citología , Fibroblastos/metabolismo , Ratas , Reproducibilidad de los Resultados , TemperaturaRESUMEN
Transcriptionally regulated growth-response genes play a pivotal role in the determination of the fate of a cell. p53 is known to transcriptionally regulate genes important in regulating cell growth potential. Using differential reverse transcription-PCR analysis of rat embryo fibroblast cells containing a temperature-sensitive p53 allele, we were able to isolate several transcripts up-regulated specifically in cells harboring functional p53 protein. Two of these genes, SM20 and microsomal epoxide hydrolase (mEH), are previously described genes. Two previously uncharacterized cDNAs, cell growth regulatory (CGR) genes CGR11 and CGR19, were isolated. The predicted amino acid sequence of these novel proteins contain known motifs; EF-hand domains (CGR11) and a ring-finger domain (CGR19), suggestive of function. CGR11 and CGR19 appear to be primary response genes expressed to moderate levels in functional p53 cells. Both CGR11 and CGR19 are able to inhibit the growth of several cell lines.
Asunto(s)
Proteínas de Unión al Calcio/genética , División Celular/genética , Regulación de la Expresión Génica , Genes Reguladores , Inhibidores de Crecimiento/genética , Proteínas/genética , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/química , ADN Complementario/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Genes p53 , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/química , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Proteínas/química , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Ratas , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Técnica de Sustracción , TemperaturaRESUMEN
The p53 tumor suppressor gene is a logical target for cancer therapy. Several therapeutic strategies can be envisioned based upon recent advances concerning structure and function of the p53 protein, its interaction with cellular and viral proteins and its roles in repairing DNA, regulating cell division and promoting apoptosis.
Asunto(s)
Genes p53/efectos de los fármacos , Neoplasias/genética , Neoplasias/terapia , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Terapia Genética , Vectores Genéticos/uso terapéutico , Humanos , Mutación , Neoplasias/inmunología , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
We report the results of a selection for single-stranded DNA oligonucleotide ligands to the serine protease thrombin using recently developed methods. This selection yielded a family of DNA sequences that conform to a consensus structure comprised of a unimolecular quadruplex motif and complementary flanking sequences capable of forming an additional Watson-Crick duplex motif. This novel quadruplex/duplex structure was not reported in a previous selection for DNA molecules which bind to thrombin [Bock et al. (1992) Nature 355, 564-566]. All quadruplex/duplex molecules tested bound to thrombin with higher affinity than quadruplex structures lacking the duplex structure. However, binding affinities did not always correlate with inhibitory potency since some molecules with high affinity were not potent inhibitors in vitro. 1H NMR spectroscopy studies demonstrated that the complementarity of bases in the duplex portion of a selected sequence allows it to form multimolecular structures. Constraining these molecules to the unimolecular quadruplex/duplex structure by bridging the 5' and 3' ends of the duplex motif with either triethylene glycol or disulfide bonds improved their thrombin inhibitory activity. All bridged quadruplex/duplex molecules were more potent inhibitors than molecules with only a quadruplex motif. Bridging the ends of these structures not only increased thrombin inhibition but also improved resistance to nucleases in serum more than 40-fold over the unbridged quadruplex. In addition, we have found that both the length and sequence of the duplex motif are important for inhibition.
Asunto(s)
ADN de Cadena Simple/química , Oligodesoxirribonucleótidos/química , Trombina/antagonistas & inhibidores , Aniones , Composición de Base , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , ADN de Cadena Simple/metabolismo , Disulfuros/química , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismo , Trombina/metabolismoRESUMEN
Salmon calcitonin (sCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. We have produced salmon calcitonin by in vitro amidation of an E. coli produced precursor peptide. Glycine-extended sCT, the substrate for amidation, was produced in recombinant E. coli as part of a fusion with glutathione-S-transferase. The microbially produced soluble fusion protein was purified to near homogeneity by affinity chromatography. Following S-sulfonation of the fusion protein, the glycine-extended peptide was cleaved from the fusion by cyanogen bromide. The S-sulfonated peptide was recovered and enzymatically converted to the amidated peptide in a reaction with recombinant peptidylglycine alpha-amidating enzyme (alpha-AE) secreted from Chinese hamster ovary (CHO) cells. After reformation of the intramolecular disulfide bond, the sCT was purified with a step yield of 60%. The ease and speed of this recombinant process, as well as its potential for scale-up, make it adaptable to production demands for calcitonin, a proven useful agent for the treatment of post-menopausal osteoporosis. Moreover, the relaxed specificity of the recombinant alpha-AE for the penultimate amino acid which is amidated allows the basic process to be applied to the production of other amidated peptides.
Asunto(s)
Calcitonina/biosíntesis , Clonación Molecular/métodos , Escherichia coli/genética , Oxigenasas de Función Mixta/metabolismo , Complejos Multienzimáticos , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Calcitonina/genética , Calcitonina/aislamiento & purificación , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Cricetinae , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/genética , Glicina , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Salmón , TransfecciónRESUMEN
Peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the conversion of glycine-extended prohormones to their biologically active alpha-amidated forms. We have derived a clonal Chinese hamster ovary cell line that secretes significant quantities of active alpha-AE. Enzyme production was increased by selection for methotrexate-resistant cells expressing a dicistronic message. Amplification of the alpha-AE gene was monitored by Southern blot analysis, enzyme activity, and immunoreactive protein throughout the selection process. The soluble enzyme is bifunctional as determined by the ability to convert either the glycine-extended substrate, dansyl-Tyr--Val--Gly, or the intermediate, dansyl-Tyr--Val--alpha-hydroxyglycine, to the dansyl-Tyr--Val--NH2 product. The recombinant alpha-AE was purified by a simple two-step chromatographic process. The purified enzyme is partially glycosylated and the glycosylated and nonglycosylated forms of the enzyme were separated on a Con A-Sepharose column. The kinetic constants for dansyl-Tyr--Val--Gly, dansyl-Tyr--Val--alpha-hydroxyglycine, ascorbate, and catechol were the same for both forms of alpha-AE. In addition, mimosine is competitive vs ascorbate with K(is) = 3.5 microM for the nonglycosylated alpha-AE and K(is) = 4.2 microM for the glycosylated alpha-AE. Therefore, the presence or absence of asparagine-linked oligosaccharide does not affect the catalytic efficiency of the enzyme. Overexpression of the recombinant enzyme in CHO cells greatly enhances expression of the endogenous gene, implicating a feedback mechanism on the alpha-AE gene.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Complejos Multienzimáticos , Secuencia de Aminoácidos , Animales , Northern Blotting , Southern Blotting , Western Blotting , Células CHO , Cromatografía por Intercambio Iónico , Células Clonales , Cricetinae , ADN/genética , ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Cinética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/aislamiento & purificación , Datos de Secuencia Molecular , Plásmidos , ARN/genética , ARN/aislamiento & purificación , Ratas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , TransfecciónRESUMEN
A rat alpha-amidating enzyme (alpha-AE) cDNA has been expressed in mouse C127 cells using a bovine papilloma virus vector in which transcription was regulated by the mouse metallothionein 1 promoter. The cDNA encoding the full length alpha-AE protein was modified to terminate translation at a site preceding the transmembrane and cytoplasmic domains, thereby enabling functional enzyme to be secreted into the medium. Purification of recombinant alpha-AE to homogeneity indicated that the enzyme was synthesized and secreted as two proteins of 75-77 kDA. The observed heterogeneity was due to inefficient glycosylation at Asn660, as demonstrated by glycopeptidase F digestion. Using the synthetic peptide, dansyl-Tyr-Val-Gly, the specific activity of the recombinant enzyme at pH 7.0 was found to be 1.4 mumol/min/mg and the Km of the enzyme was determined to be 3 microM. The purified recombinant enzyme has maximal activity at pH 4.5-5.5; however, a rapid inactivation of the enzyme occurs in acidic solutions in vitro. This inactivation is diminished when activity is measured at pH 7.0-10.0. The availability of large amounts of readily purified, active recombinant alpha-AE should allow detailed probing of reaction mechanism, copper coordination chemistry, and turn-over-based inactivation events.
Asunto(s)
Oxigenasas de Función Mixta/aislamiento & purificación , Complejos Multienzimáticos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Vectores Genéticos , Cinética , Ratones , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sondas de Oligonucleótidos , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The alpha-amidating enzyme activity in rat medullary thyroid carcinoma (MTC) consists of multiple, active enzymes that can be resolved by ion-exchange chromatography. Amino acid sequences from one form of purified rat MTC alpha-amidating enzyme have been utilized to design oligonucleotide probes for isolating cDNAs encoding this protein. Sequence analysis of multiple cDNA clones indicates that there are at least two types of cDNA in rat tissues. These cDNAs differ primarily by the absence (type A) or the presence (type B) of a 315-base internal sequence. Additional heterogeneity in the 3' coding regions of the different mRNAs has also been found. Both types of cDNA predict primary translation products that are preproenzymes which must be post-translationally processed at both their amino and carboxyl termini. Sequence analysis of the purified peak III protein from rat MTC demonstrates that the type A mRNA encodes this 75-kDa protein. This analysis also provides support for the assignment of the C-terminal processing site. In addition, data are presented which demonstrate that type B mRNA is also functional. The implications of the internal and carboxyl-end heterogeneity are discussed.
Asunto(s)
Carcinoma/genética , ADN/análisis , Oxigenasas de Función Mixta , Complejos Multienzimáticos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , ARN Mensajero/metabolismo , Neoplasias de la Tiroides/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carcinoma/enzimología , Clonación Molecular , Datos de Secuencia Molecular , Peso Molecular , Empalme del ARN , Ratas , Mapeo Restrictivo , Neoplasias de la Tiroides/enzimología , Células Tumorales CultivadasRESUMEN
The biosynthesis and secretion of alpha amidating enzymes from CA-77 cells has been investigated to determine the relationship among the various forms of alpha amidating enzyme seen after purification of alpha amidating enzyme activity from conditioned cell culture media. Initially 2 proteins of 104 kD and 94 kD are synthesized. With time the 104 kD precursor is processed to 41 kD and 43 kD, and the 94 kD precursor is processed to 75 kD. The 41 kD, 43 kD, and 75 kD proteins are secreted into the medium as functional enzymes. In comparing these data with known cDNA sequence for alpha amidating enzyme we conclude that the 104 kD and 94 kD precursors are membrane bound proteins which are posttranslationally processed to generate secreted alpha amidating enzyme.
Asunto(s)
Precursores Enzimáticos/metabolismo , Oxigenasas de Función Mixta , Complejos Multienzimáticos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Línea Celular , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Glicosilación , Hidrólisis , Microsomas/metabolismo , ARN Mensajero/metabolismo , Ratas , Espectrometría de FluorescenciaRESUMEN
Conditioned medium from cultures of rat medullary thyroid carcinoma CA-77 cells was used as a source for purification of the secreted forms of peptidyl alpha-amidating enzyme. The alpha-amidating enzyme activity was partially purified using a combination of weak anion exchange and gel filtration chromatography. Subsequent strong anion exchange chromatography at pH 6.0 resolved this partially pure enzyme into four distinct peaks of activity, termed Ia, Ib, II, and III. Peaks Ia and Ib exhibited broad pH optima between pH 6.0-8.5, whereas peaks II and III both exhibited pH optima at approximately pH 5.0. The peak III activity was further purified to electrophoretic homogeneity using hydrophobic interactive chromatography followed by strong anion exchange chromatography at pH 8.0. The enzyme exhibited an apparent molecular mass of 75K, a pH optimum of approximately pH 5.0, and a maximal turnover number of 580 min-1 in the presence of L-ascorbate. Kinetic studies demonstrated that the enzyme probably functions through a ping-pong mechanism with respect to the binding of the glycine-extended peptide substrate and the L-ascorbate cofactor. The peak III enzyme exhibits several distinctive characteristics compared to amidating enzymes isolated and characterized by other laboratories.
Asunto(s)
Isoenzimas/metabolismo , Oxigenasas de Función Mixta , Complejos Multienzimáticos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Neoplasias de la Tiroides/enzimología , Animales , Línea Celular , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Medios de Cultivo , Isoenzimas/aislamiento & purificación , Cinética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/aislamiento & purificación , RatasRESUMEN
Posttranslational carboxyl-terminal amidation of many peptides is accomplished by peptidylglycine alpha-amidating monooxygenase. We have previously demonstrated that glucocorticoids stimulate production of amidated products by the CA-77 rat medullary thyroid carcinoma cell line. The present investigation was undertaken to determine whether amidation enzyme activity changes in parallel. Enzyme activity, similar to that found in other tissues, was readily detected in cell extracts and conditioned cultured medium. Stimulation with the calcitonin secretagogue calcium increased secretion of enzyme activity and lowered cell extract activity. Treatment of cultures with dexamethasone, but no other steroid, decreased by 50-70% the basal amidation enzyme activity secreted. There was no associated change in cellular activity. The decrease in medium activity was partially reversible and steroid-dose dependent. The glucocorticoid-induced change in medium activity was due to a decreased Vm. These experiments demonstrate that the alpha-amidating activity of the CA-77 cells can be hormonally regulated.
Asunto(s)
Dexametasona/farmacología , Oxigenasas de Función Mixta , Complejos Multienzimáticos , Neoplasias Experimentales/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/análisis , Animales , Medios de Cultivo , Activación Enzimática/efectos de los fármacos , Concentración de Iones de Hidrógeno , Proteínas de Neoplasias/metabolismo , Ratas , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
A peptidyl glycine alpha-amidating activity has been isolated from total tissue extracts of rat medullary thyroid carcinoma (MTC). Purification of the activity by ammonium sulfate fractionation, Sephacryl S-300 chromatography, and strong anion-exchange chromatography at pH 6.0 has resolved at least four peaks of activity. The activity associated with peak III has been further purified to apparent homogeneity by strong anion-exchange chromatography at pH 8.0. The purified peak III enzyme has an apparent molecular mass of 75,000 Da as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of the 75,000-Da band as the alpha-amidating enzyme has been confirmed by recovery of activity from a nondenaturing polyacrylamide gel. The enzyme is catalytically active as a monomer, exhibits a pH optimum between 5.0 and 5.5, and has a turnover number of 300 min-1 for N-dansyl-Tyr-Val-Gly amidation at pH 5.5. The larger size, more acidic pH optimum, and higher specific activity distinguish the purified peak III rat MTC enzyme from the enzymes isolated from bovine and porcine pituitary or from frog skin.
Asunto(s)
Oxigenasas de Función Mixta , Complejos Multienzimáticos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/aislamiento & purificación , Neoplasias de la Tiroides/enzimología , Animales , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cinética , Peso Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , RatasRESUMEN
Several size classes of small polydisperse circular (spc) DNA from the African green monkey cell line BSC1 have been cloned into the bacterial plasmid pBR322. Analysis of the cloned spc DNA fragments as well as total spc DNA reveals that (a) most or all cloned spc DNAs share homologies with chromosomal sequences, (b) both unique and repetitive chromosomal sequences are represented in spc DNA, (c) the repetitive sequences in spc DNA include two known major repeat families (the alpha and the Alu) as well as a third, as yet unidentified, set of interspersed repetitive sequences, and (d) the alpha-like sequences are present in an oligomeric series of circular DNA molecules within the spc DNA population. The organizational features of repetitive DNA sequence-carrying circles suggest a mechanism for their generation.
Asunto(s)
ADN Circular/aislamiento & purificación , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Cromosomas , Clonación Molecular , Hibridación de Ácido Nucleico , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The stable uptake of a bacterial plasmid by mouse or rat cells has been detected following the respective co-transfection of these cells with plasmid in the presence of either Simian virus 40 (SV40) or Polyoma virus DNAs. About 60-70 percent of the resulting SV40-transformed mouse cell clones and about 40 percent of the Polyoma transformed rat cell clones contained plasmid DNA sequences.