RESUMEN

AIMS: The aim of this study was to investigate associations between coronary heart disease risk and polymorphisms in the coagulation factor (F)VII gene in participants of a large prospective study. METHODS: One thousand nine hundred and fifty-seven men were genotyped for four FVII polymorphisms, -670A-->C, -402G-->A, a 10 base pair insertion at -323 (0 > 10) in the promoter, and R353Q in the structural gene. Associations among genotypes and estimated haplotypes, plasma FVII levels, and coronary heart disease risk were evaluated, and the function of the promoter polymorphisms was assessed in reporter gene assays. RESULTS: The -670A-->C and -402G-->A polymorphisms were in complete allelic association. The haplotype containing -670C and -402A (frequency =0.23) was associated with significantly increased plasma FVII coagulant activity and increased risk of an initial coronary event, particularly acute myocardial infarction, which remained after correction for conventional risk factors. In contrast, the -323 insertion and Q353 alleles (frequency =0.11 and 0.10, respectively) were associated with decreased plasma FVII levels, but hazard ratios for coronary events in carriers of these alleles were not significantly different from unity. In transiently transfected hepatoma cells, increased basal expression of the reporter gene was directed by a promoter fragment with rare haplotype -670C/-630G/-402A rather than by a promoter fragment with common haplotype -670A/-630A/-402G; -402A was not responsible for this effect. CONCLUSIONS: The promoter haplotype, -670C/-630G/402A, was associated with significantly increased plasma FVII coagulant activity, risk of an initial coronary event, particularly acute myocardial infarction, and reporter gene expression.

Asunto(s)

Enfermedad Coronaria/genética , Factor VII/genética , Alelos , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Exones , Factor VII/biosíntesis , Genes Reporteros , Genotipo , Haplotipos , Humanos , Intrones , Masculino , Persona de Mediana Edad , Infarto del Miocardio/genética , Polimorfismo Genético , Regiones Promotoras Genéticas , Modelos de Riesgos Proporcionales , Riesgo , Transfección

RESUMEN

Factor (F)VII plays a critical role in initiation of coagulation. Several segments within the 5' flanking region of the FVII gene were previously demonstrated to recognize hepatic nuclear proteins, but few have been identified. To identify a regulatory protein binding the nuclear hormone response region (-237 to -200) of the FVII 5' flanking region and demonstrate that the interaction is functional. Electrophoretic mobility shift assays and mutation analysis showed that ARP1, an orphan nuclear hormone receptor, interacted with two regions of the FVII 5' flanking region, the hepatic nuclear factor 4 binding region (-77 to -47) and the nuclear hormone response region (-237 to -200). Transfection experiments demonstrated that reporter gene expression was decreased from vectors including the nuclear hormone response segment compared with that containing only the minimal promoter between positions -109 and +1, and that ARP1 also repressed expression through an interaction with the minimal promoter. These data indicate a role for ARP1 in transcriptional modulation of the FVII gene.

Asunto(s)

Región de Flanqueo 5'/genética , Proteínas de Unión al ADN/metabolismo , Factor VII/genética , Receptores de Esteroides , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión , Factor de Transcripción COUP II , Factores de Transcripción COUP , Línea Celular Tumoral , Proteínas de Unión al ADN/fisiología , Genes Reporteros , Factor Nuclear 4 del Hepatocito , Humanos , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/genética , Elementos de Respuesta , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transfección

RESUMEN

A case of a novel mutation in the F7 gene that results in factor VII coagulant activity (VII:c) of less than 1% and VII antigen (VII:Ag) levels of 10% is presented. DNA analysis revealed a homozygous 15-base pair (bp) in-frame insertion-type mutation at nucleotide 10554. This insertion consisted of a duplication of residues leucine (L)213 to aspartic acid (D)217 (leucine, serine, glutamic acid, histidine, and aspartic acid), probably arising by slipped mispairing between 2 copies of a direct repeat (GCGAGCACGAC) separated by 4 bp. Molecular graphic analyses showed that the insertion is located at the surface of the catalytic domain in an exposed loop stabilized by extensive salt-bridge and hydrogen bond formation at which the calcium binding site is located. The mutation probably interferes with protein folding during VII biosynthesis and/or diminishes functional activity through the loss of calcium binding. In vitro expression studies demonstrated that the levels of VII:Ag in lysates of cells transfected with wild type VII (VIIWT) were equivalent to those with mutant type VII (VIIMT), but the level of secreted VIIMT was 5% to 10% that of VIIWT. Pulse chase studies demonstrated that VIIMT did not accumulate intracellularly, and studies with inhibitors of protein degradation showed that recombinant VIIMT was partially degraded in the pre-Golgi compartment. Accordingly, only small amounts of VIIMT with undetectable procoagulant activity were secreted into conditioned media. These results demonstrate that a combination of secretion and functional defects is the mechanism whereby this insertion causes VII deficiency.

Asunto(s)

Deficiencia del Factor VII/genética , Factor VII/genética , Mutagénesis Insercional , Disparidad de Par Base , Sitios de Unión , Calcio/metabolismo , Preescolar , Clonación Molecular , Consanguinidad , Análisis Mutacional de ADN , Factor VII/biosíntesis , Factor VII/química , Factor VII/metabolismo , Femenino , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Relación Estructura-Actividad

RESUMEN

Investigation of the molecular basis of a severe factor VII (fVII) deficiency revealed compound heterozygosity in the fVII gene. On the paternal allele the patient had 3 structural gene abnormalities frequently associated with fVII deficiency. A new mutation, a C to T transition at position -55 relative to the translational start site, was found on the maternal allele. The study demonstrates that this mutation partially impeded binding of the transcriptional activator, hepatic nuclear factor 4, to the fVII promoter while greatly reducing reporter gene expression in hepatic cells. (Blood. 2000;96:4370-4372)

Asunto(s)

Proteínas de Unión al ADN , Deficiencia del Factor VII/genética , Factor VII/genética , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Adulto , Sustitución de Aminoácidos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión , Unión Competitiva , Células Cultivadas , Codón/genética , Análisis Mutacional de ADN , Mutación del Sistema de Lectura , Genotipo , Factor Nuclear 4 del Hepatocito , Heterocigoto , Humanos , Masculino , Oligodesoxirribonucleótidos/metabolismo , Mutación Puntual , Polonia , Proteínas Recombinantes/metabolismo , Transcripción Genética , Transfección

RESUMEN

We investigated a Sephardic Jewish patient with a mild bleeding diathesis whose plasma levels of factor VII coagulant activity and factor VII antigen were 7% and 9% of normal, respectively. Sequencing demonstrated homozygosity for the Ala244Val mutation and the Arg353Gln polymorphism, which is associated with a modest decrease in factor VII levels. To elucidate the mechanism by which Ala244Val reduced factor VII levels in this patient, transient transfections were performed in COS-1 cells with wild type and mutant factor VII cDNAs and factor VII antigen levels in cell lysates and conditioned media were measured. The secretion of the mutant protein (FVII244V) into the media was 20% of wild type (FVIIwt), and intracellular levels of FVII244V were 60% of FVIIwt. A construct encoding Ala244Val along with the Arg353Gln polymorphism decreased the factor VII level in the media to that observed in the patient's plasma. Pulse-chase experiments demonstrated that FVII244V did not accumulate intracellularly and that low levels of the abnormal protein were maintained throughout the chase. To test the hypothesis that FVII244V results in an unstable molecule, amino acids with smaller (Gly) or larger (Phe) side chains were substituted for Val244 by site-directed mutagenesis. Transient transfection assays with these constructs demonstrated that the side chain of amino acid 244 is crucial in maintaining a proper conformation of the molecule. We conclude that Ala244Val results in a factor VII molecule that is unstable and is probably degraded intracellularly.

Asunto(s)

Deficiencia del Factor VII/genética , Mutación Missense/genética , Alanina/genética , Factor VII/biosíntesis , Deficiencia del Factor VII/etnología , Femenino , Homocigoto , Humanos , Judíos , Masculino , Persona de Mediana Edad , Linaje , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Valina/genética

RESUMEN

We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys97 did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII-Arg100, the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg100 was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII-Arg100 had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation.

Asunto(s)

Deficiencia del Factor VII/genética , Factor VII/genética , Mutación , Animales , Células CHO , Cricetinae , Factor de Crecimiento Epidérmico/genética , Factor VII/química , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Italia

RESUMEN

We have identified a point mutation in the promoter of the factor VII gene responsible for a severe bleeding disorder in a patient from a large French-Canadian family with known consanguinity. The proband has an extremely low plasma level of factor VII antigen and factor VII coagulant activity (<1 percent of normal) and suffers from hemarthroses and chronic arthropathy. Sequencing of the patient's factor VII 5' flanking region, intron/exon junctions, and coding regions showed a homozygous point mutation, a C to G transversion at position -94 relative to the translation start site. We show here that this mutation prevented binding of transcription factor Sp1 and of other nuclear proteins to this region of the factor VII promoter and resulted in a 20-fold reduction in reporter gene expression in HepG2 cells. These data underscore the importance of this region of the factor VII promoter for in vivo expression of the factor VII gene.

Asunto(s)

Deficiencia del Factor VII/genética , Factor VII/genética , Mutación , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Adulto , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Consanguinidad , ADN/química , ADN/metabolismo , Femenino , Células HeLa/ultraestructura , Humanos , Masculino

RESUMEN

Factor VII levels are regulated by environmental and genetic factors. Two polymorphisms, a G-to-A transversion at nucleotide 10,976 resulting in Arg353Gln and a decanucleotide insert at position -323 in the 5'-flanking region of the factor VII gene, have been associated with a 20% to 25% reduction in plasma factor VII levels. However Arg353Gln almost always segregates on alleles containing the insert in UK and Italian populations, thereby making it impossible to independently evaluate the impact of Arg353Gln on factor VII levels in these ethnic groups. We have evaluated the influence of genotype on factor VII levels in 99 healthy Polish blood donors and observed that Arg353Gln frequently occurs in the absence of the insert. In univariate analysis, the mean levels of factor VII coagulant activity (VII:C) and factor VII antigen (VII:Ag) were significantly lower in 16 people who were heterozygous for Arg353Gln and the insert compared with 72 normal subjects who had neither Arg353Gln nor the insert (88.8% of normal and 83.1% versus 102% and 100%, P = .019 and P = .0003, respectively). In nine subjects heterozygous for Arg353Gln alone, VII:C and VII:Ag were significantly decreased compared with the normal subjects (81.9% and 83%, respectively, P = .007 and P = .004). In multivariate analysis, Arg353Gln but not the insert significantly reduced VII:C and VII:Ag after adjustment for age and plasma triglycerides (P < .05 and P = .02, respectively). To evaluate the mechanism responsible for reduced factor VII levels in individuals with Arg353Gln, we performed transient transfection assays with factor VII cDNA containing the base substitution resulting in Gln353 and wild-type factor VII cDNA in COS-1 cells. The levels of VII:Ag in the cell lysates were similar, but the amino acid substitution significantly reduced factor VII secretion into the media to 74.9% of wild-type (P = .0001). Based on these in vivo and in vitro studies, we conclude that the Arg353Gln polymorphism alone can decrease plasma factor VII levels.

Asunto(s)

Deficiencia del Factor VII/genética , Factor VII/genética , Mutación Puntual , Polimorfismo Genético , Animales , Células CHO , Células COS , Células Cultivadas , Codón/genética , Cricetinae , Cricetulus , Análisis Mutacional de ADN , ADN Complementario/genética , Deficiencia del Factor VII/sangre , Factor VIIa/análisis , Genotipo , Humanos , Mutagénesis Insercional , Polonia , Transfección , Triglicéridos/sangre

RESUMEN

We show here that NFAT1 is rapidly activated, then slowly deactivated, by stimulation of T cells through their antigen receptor. Within minutes of T-cell receptor stimulation, NFAT1 is dephosphorylated, translocates from the cytoplasm into the nucleus, and shows an increase in its ability to bind to DNA. These changes are dependent on calcium mobilization and calcineurin activation, since they are also elicited by ionomycin and are blocked by the immunosuppressive drug cyclosporin A. After several hours of T-cell receptor stimulation, the majority of the NFAT1 in the cell reverts to its original phosphorylated form, reappears in the cytoplasm, and again displays a low affinity for DNA. Deactivation of NFAT1 is facilitated by phorbol 12-myristate 13-acetate and inhibitors of capacitative calcium entry and most likely reflects the slow return of intracellular free calcium concentrations towards resting levels. Our results suggest that calcineurin-dependent signalling pathways mediate the early activation of NFAT1, while phorbol 12-myristate 13-acetate-dependent feedback pathways contribute to the late deactivation. Persistent NFAT-dependent cytokine gene transcription in activated T cells may be mediated by other NFAT family proteins in addition to NFAT1 during the immune response.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Activación de Linfocitos , Proteínas Nucleares , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Factores de Transcripción/metabolismo , Animales , Células CHO , Núcleo Celular/metabolismo , Células Clonales , Cricetinae , Citoplasma/metabolismo , Antígenos de Histocompatibilidad Clase II/biosíntesis , Ionomicina/farmacología , Ratones , Factores de Transcripción NFATC , Fosfoproteínas/metabolismo , Proteínas Recombinantes/biosíntesis , Transfección

RESUMEN

Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. We have identified two new isoforms of the transcription factor NFAT1 (previously termed NFATp) that are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive agent cyclosporin A (CsA). Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol 12-myristate 13-acetate; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol 12-myristate 13-acetate. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response.

Asunto(s)

Citocinas/biosíntesis , Proteínas de Unión al ADN/metabolismo , Linfocitos T/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Chlorocebus aethiops , Clonación Molecular , Cartilla de ADN , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

By transfecting the full-length cDNA for human von Willebrand factor (vWf) into a line of Chinese hamster ovary cells with a defect in carbohydrate metabolism, we have prepared recombinant vWf specifically lacking O-linked carbohydrates. We have compared this under-glycosylated protein to fully glycosylated recombinant vWf with respect to several structural and binding properties. vWf deficient in O-linked glycans was synthesized, assembled into multimers, and secreted in an apparently normal manner and was not prone to degradation in the extracellular milieu. It did not differ from fully glycosylated vWf in ability to bind to heparin or to collagen type I but did interact less well with glycoprotein 1b on formalin-fixed platelets. This decreased interaction was evidenced in both a lessened overall binding to platelets and in diminished capacity to promote platelet agglutination, in the presence of ristocetin. In contrast, no difference was seen in platelet binding in the presence of botrocetin. These data indicate a possible role for O-linked carbohydrates in the vWf-glycoprotein 1b interaction promoted by ristocetin and suggest that abnormalities in carbohydrate modification might contribute to the altered ristocetin-dependent reactivity between vWf and platelets described for some variant forms of von Willebrand disease.

Asunto(s)

Glicoproteínas de Membrana Plaquetaria/metabolismo , Ristocetina/farmacología , Factor de von Willebrand/metabolismo , Secuencia de Aminoácidos , Colágeno/farmacología , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Heparina/farmacología , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Datos de Secuencia Molecular , Agregación Plaquetaria , Unión Proteica , Proteínas Recombinantes , Relación Estructura-Actividad , Factor de von Willebrand/química

RESUMEN

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein essential for normal hemostasis. We have discovered that cultured human umbilical vein endothelial cells incorporate inorganic sulfate into vWF. Following immunoisolation and analysis by polyacrylamide or agarose gel electrophoresis, metabolically labeled vWF was found to have incorporated [35S]-sulfate into all secreted multimer species. The time course of incorporation shows that sulfation occurs late in the biosynthesis of vWF, near the point at which multimerization occurs. Quantitative analysis suggests the presence, on average, of one molecule of sulfate per mature vWF subunit. Virtually all the detectable sulfate is released from the mature vWF subunit by treatment with endoglycosidases that remove asparagine-linked carbohydrates. Sulfated carbohydrate was localized first to the N-terminal half of the mature subunit (amino acids 1 through 1,365) by partial proteolytic digestion with protease V8; and subsequently to a smaller fragment within this region (amino acids 273 through 511) by sequential digestions with protease V8 and trypsin. Thus, the carbohydrate at asparagine 384 and/or 468 appears to be the site of sulfate modification. Sodium chlorate, an inhibitor of adenosine triphosphate-sulfurylase, blocks sulfation of vWF without affecting either the ability of vWF to assemble into high molecular weight multimers or the ability of vWF multimers to enter Weible-Palade bodies. The stability of vWF multimers in the presence of an endothelial cell monolayer also was unaffected by the sulfation state. Additionally, we have found that the cleaved propeptide of vWF is sulfated on asparagine-linked carbohydrate.

Asunto(s)

Sulfatos/metabolismo , Factor de von Willebrand/metabolismo , Cloratos/farmacología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Herbicidas/farmacología , Humanos , Mapeo Peptídico , Serina Endopeptidasas/farmacología , Radioisótopos de Azufre , Tripsina/farmacología , Factor de von Willebrand/análisis

RESUMEN

Untransformed and malignantly-transformed mouse embryo fibroblasts were found to contain an enzymatic activity which hydrolysed the synthetic substrate, Suc-Ala-Ala-Pro-Phe-AMC. This activity, of approximate molecular weight 55,000, which has been partially purified by ion-exchange and gel-filtration chromatography, was maximally active at neutral pH, associated with subcellular organelles or membranes and inhibited by EDTA, EGTA, phosphoramidon and 1,10 phenanthroline, but not by PMSF or pepstatin, indicating that it may be a metalloprotease. Several other protease inhibitors, such as chymostatin, TPCK and the BBI from soybean, were also potent inhibitors of the activity; these same inhibitors are known to suppress the malignant transformation of mouse embryo fibroblast cells in vitro induced by X-irradiation or chemical carcinogens. In contrast, SBTI and pepstatin, which did not inhibit this activity, also do not suppress malignant transformation. These results suggest that this enzyme may be involved in attainment of the transformed state.

Asunto(s)

Transformación Celular Neoplásica/efectos de los fármacos , Metaloendopeptidasas/metabolismo , Inhibidores de Proteasas/farmacología , Animales , Línea Celular , Transformación Celular Neoplásica/metabolismo , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Fibroblastos/enzimología , Concentración de Iones de Hidrógeno , Hidrólisis , Peso Molecular , Fracciones Subcelulares/enzimología , Especificidad por Sustrato

RESUMEN

In the present study the effect of feeding the soybean-derived Bowman-Birk protease inhibitor (BBI) on dimethylhydrazine (DHM)-induced gastrointestinal tract and liver carcinogenesis in mice was examined. In this investigation we found the addition of 0.5 or 0.1% semipurified BBI or 0.1% purified BBI to the diet of DMH-treated mice resulted in a statistically significant suppression of angiosarcomas and nodular hyperplasia of the liver and adenomatous tumors of the gastrointestinal tract. Autoclaved BBI or BBI which had its trypsin inhibitory domain specifically inactivated was found to be ineffective in suppressing the induction of these liver and gastrointestinal tract lesions. The results of this study also indicate that BBI, included as 0.5% of the diet or less, has the ability to suppress carcinogenesis with no observed adverse effects on the health of the mice.

Asunto(s)

Dimetilhidrazinas , Metilhidrazinas , Neoplasias Experimentales/inducido químicamente , Inhibidor de la Tripsina de Soja de Bowman-Birk/administración & dosificación , Inhibidores de Tripsina/administración & dosificación , Adenocarcinoma/inducido químicamente , Animales , Peso Corporal/efectos de los fármacos , Carcinoma de Células Escamosas/inducido químicamente , Neoplasias Gastrointestinales/inducido químicamente , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas Experimentales/inducido químicamente , Ratones , Ratones Endogámicos , Tamaño de los Órganos/efectos de los fármacos , Páncreas/anatomía & histología

RESUMEN

Bacterial endotoxins (lipopolysaccharides) were found to suppress X-ray-induced malignant transformation of C3H/10T1/2 cells. Endotoxins were effective if present either throughout the 6-week transformation assay period, or for the final 4-week phase, but not when present only for the initial 2-week phase. Neither growth nor survival of C3H/10T1/2 cells, or a radiation-transformed cell line derived from them, were affected by endotoxins. Also, the endotoxins did not affect the formation of foci by the radiation transformed cells when these cells were co-cultured with untransformed cells. These results suggest that endotoxins exert their effect directly upon the transformation process itself, perhaps at a 'late' step in the conversion of an untransformed to a transformed cell.

Asunto(s)

Transformación Celular Neoplásica/efectos de la radiación , Endotoxinas/farmacología , Animales , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ratones

RESUMEN

Several different protease inhibitors have the ability to suppress transformation in vitro and carcinogenesis in vivo. The mechanism(s) by which protease inhibitors suppress carcinogenesis, however, is not fully understood. Presumably, these agents inhibit one or more intracellular proteases whose functions are essential for the induction and/or expression of the transformed phenotype. We have isolated an endopeptidase activity capable of hydrolyzing the substrate Boc-Val-Pro-Arg-MCA (Boc = butoxycarbonyl; MCA = 7-amino-4-methylcoumarin) from C3H/10T1/2 mouse embryo fibroblast cells. This intracellular protease was inhibited by the soybean-derived Bowman-Birk inhibitor (BBI), chymostatin, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, all of which have anticarcinogenic activity, but was unaffected by soybean trypsin inhibitor, which lacks anticarcinogenic activity. Other protease inhibitors affected the proteolytic activity to an extent that correlates with their relative ability to suppress transformation in vitro. The enzyme has a mass of about 70 kDa, contains a single subunit, and exhibits maximal activity at pH 7.0. Diisopropyl fluorophosphate covalently binds to this enzyme and blocks its activity, indicating that the enzyme is a serine protease. We have previously demonstrated that several protease inhibitors are effective suppressors of radiation-induced transformation of C3H/10T1/2 cells. Since these agents reduce the Boc-Val-Pro-Arg-MCA-hydrolyzing activity to an extent that correlates with their ability to inhibit malignant transformation in vitro, this endopeptidase activity may be a cellular target of the anticarcinogenic protease inhibitors.

Asunto(s)

Endopeptidasas/aislamiento & purificación , Fibroblastos/enzimología , Inhibidores de Proteasas/farmacología , Proteínas/farmacología , Animales , Línea Celular , Transformación Celular Neoplásica/prevención & control , Cumarinas/metabolismo , Endopeptidasas/metabolismo , Isoflurofato/farmacología , Ratones , Ratones Endogámicos C3H , Oligopéptidos/metabolismo , Serina Endopeptidasas , Inhibidores de Serina Proteinasa

RESUMEN

A human damage-specific DNA binding protein has been employed as a sensitive probe of damage introduction by the combination of Cu(II) and H2O2. Optimal conditions for the introduction of protein-recognizable lesions into DNA in the Cu(II)/H2O2 system were obtained with 10(-5)-M CuCl2 and 0.10-mM H2O2. The absolute requirement for the presence of a metal ion suggests the involvement of a metal catalyzed Fenton reaction. However, damage introduction in the presence of KI and dimethylsulfoxide indicate that hydroxyl radical, while responsible for the introduction of strand breaks, is not the primary species responsible for lesion introduction. Protein-recognizable damage was introduced into DNA and poly d(G-C), but not into poly d(A-T). Loss of label from the five position of cytosine was also observed at high peroxide levels.

Asunto(s)

Secuencia de Bases/efectos de los fármacos , Cobre/farmacología , Proteínas de Unión al ADN , ADN , Peróxido de Hidrógeno/farmacología , Secuencia de Bases/efectos de la radiación , Centrifugación por Gradiente de Densidad , Proteínas de Unión al ADN/análisis , Dimetilsulfóxido/farmacología , Radicales Libres , Humanos , Oxígeno/farmacología , Rayos Ultravioleta

RESUMEN

Sodium bisulfite reacts with cytosine and 5-methylcytosine, forming the 5,6-dihydrosulfonate adducts which deaminate to the uracil and thymine adducts, respectively. At alkaline pH, the sulfonate groups are then released, generating uracil and thymine. In DNA, the resulting G:U and G:T base mismatches generated are potential sites of mutagenesis. Using a human damage-specific DNA binding protein as a probe, we have found protein-recognizable lesions in bisulfite-treated DNA and poly d(I-C), but not in treated poly d(A-T) or poly d(A-U). Although this suggests that the lesion recognized is cytosine-derived, there was no correlation between the number of uracils induced and the number of binding sites, suggesting that the protein-bound damage is not a uracil-containing mismatch. Modification of the treatment protocol to reduce elimination of the bisulfite from the base adducts increased the level of binding, suggesting that the protein recognizes a base-sulfonate adduct.

Asunto(s)

Citosina/metabolismo , ADN Glicosilasas , Proteínas de Unión al ADN/fisiología , Mutación , Reparación del ADN , Humanos , Concentración de Iones de Hidrógeno , N-Glicosil Hidrolasas/metabolismo , Sulfitos/metabolismo , Temperatura , Uracilo/metabolismo , Uracil-ADN Glicosidasa

Asunto(s)

ADN Helicasas/efectos de la radiación , ADN/efectos de la radiación , Conformación de Ácido Nucleico/efectos de la radiación , Proteínas de Unión al ADN , Humanos , Fotólisis , Polidesoxirribonucleótidos/efectos de la radiación , Unión Proteica

RESUMEN

The radiation chemistry of nucleotide damage introduction into DNA gamma-irradiated in dilute aqueous solution has been studied with a damage-specific DNA binding protein. Irradiation in air, N2 or N2O in the presence and absence of free radical scavengers revealed that protein-recognizable lesions were introduced both by the hydrated electron and by the combination of the hydroxyl radical and superoxide anion radical, but not by the hydroxyl radical alone. In addition, nucleotide damage was introduced into DNA by an enzymatic superoxide-generating system.

Asunto(s)

ADN Viral/efectos de la radiación , NADPH Oxidasas , Nucleótidos/efectos de la radiación , Agua/efectos de la radiación , Proteínas Portadoras/farmacología , ADN/farmacología , Reparación del ADN , Proteínas de Unión al ADN , Radicales Libres , Rayos gamma , Humanos , Hidróxidos , Radical Hidroxilo , Técnicas In Vitro , NADH NADPH Oxidorreductasas/farmacología , Superóxidos/farmacología , Fagos T