RESUMEN

Chromatin condensation and DNA cleavage at internucleosomal sites have been recognized early as hallmarks of apoptosis, and it has been suggested that extensive DNA chain scission could directly result in the formation of dense chromatin bodies. Here we have shown that no causal relationship exists between DNA degradation and chromatin condensation in glucocorticoid-induced thymocyte apoptosis. The chromatin rearrangement occurred independent of as well as prior to DNA cleavage and involved a specific conformational change at the nucleosome level. In the early stages of the process, the core particles appeared to be tightly packed face-to-face in smooth 11-nm filaments that progressively folded to generate a closely woven network. The network finally collapsed, producing dense apoptotic bodies. Since trypsin digestion relaxed condensed chromatin and histone H4 underwent appreciable deacetylation in the apoptotic cell, we suggest that changes in the DNA-histone interactions represented a major modulating factor of condensation.

Asunto(s)

Apoptosis , Cromatina/ultraestructura , ADN/ultraestructura , Linfocitos T/fisiología , Linfocitos T/ultraestructura , Animales , Cromatina/efectos de los fármacos , ADN/efectos de los fármacos , Glucocorticoides/farmacología , Histonas/metabolismo , Hemisuccinato de Metilprednisolona/farmacología , Microscopía Electrónica , Conformación de Ácido Nucleico , Nucleosomas/efectos de los fármacos , Nucleosomas/ultraestructura , Ratas , Linfocitos T/efectos de los fármacos

Asunto(s)

Procesamiento de Imagen Asistido por Computador/métodos , Queratinas/metabolismo , Neoplasias Hepáticas/metabolismo , Animales , Inmunohistoquímica , Neoplasias Hepáticas/patología , Masculino , Ratas , Ratas Endogámicas F344

RESUMEN

We have characterized the changes in composition of the nuclear matrix-intermediate filament complex (NM-IF) isolated from prostate cancer (PCa), compared with benign prostatic hyperplasia (BPH). We prepared the NM-IF from ten patients undergoing radical retropubic prostectomy; the benign hyperplastic tissue was obtained from the prostate lobe contralateral to the cancer zone. Several quantitative and qualitative changes have been identified. Three new proteins of molecular weight 48, 47 and 29 kDa and isoelectric point 6.0, 4.9 and 6.4, respectively, were detected in PCa, referred to here as P8, P5 and NM-1, P8 was present in all ten of the tumors examined, P5 was expressed in 9/10 PCa; conversely, they were present in only one and two BPH, respectively; NM-1 was found in eight tumors out of nine and never in BPH. These proteins are expressed in moderately differentiated malignant cells, suggesting that the proteins of the NM-IF complex can be interesting biomarkers for prostate cancer. Immunoblot analysis shows that P8 and P5 proteins belong to the IF superfamily. This observation, taken together with previous data obtained by our and other groups, suggests that the characterization of NM-IF protein changes could also shed light on mechanistic aspects of cancer progression.

Asunto(s)

Proteínas de Filamentos Intermediarios/análisis , Proteínas de Neoplasias/análisis , Matriz Nuclear/química , Proteínas Nucleares/análisis , Próstata/química , Hiperplasia Prostática , Neoplasias de la Próstata/química , Anciano , Antígenos Nucleares , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

Regulation of proliferation and migration are well known roles of fibroblast growth factor 2 (FGF-2) for arterial smooth muscle cells (SMC). We show here, by sense cDNA transfection that endogenous FGF-2 expression controls alpha-actin level in SMC clones. All the high alpha-actin expressing clones were FGF-2 transfected. Control clones carrying a deleted vector showed a weak expression and an altered actin polymerisation compared to the parental cultures. Among FGF-2 transfected clones, alpha-actin expression was heterogenous with diversely high levels. These observations were obtained using normal rat SMC or SMC from a transformed cell line. They indicate a role for endogenous FGF-2 in arterial SMC differentiation. Our results suggest that FGF-2 might act either by permissing clonal growth of already differentiated cells or by regulating expression or stability of alpha-actin. They open new perspective for gene therapy of the arterial wall.

Asunto(s)

Actinas/análisis , Factor 2 de Crecimiento de Fibroblastos/genética , Músculo Liso Vascular/química , Transfección , Animales , Diferenciación Celular , Células Clonales/química , Expresión Génica , Vectores Genéticos , Músculo Liso Vascular/citología , Ratas , Ratas Wistar , Transgenes

RESUMEN

In a previous paper (Barboro et al., 1993, Biophys. J. 65, 1690-1699) we have shown that cancer development in the resistant hepatocyte model of Solt and Farber is characterized by the progressive unfolding of the higher-order structure of chromatin. A possible functional role of decondensation phenomena in cell transformation cannot be ruled out. Genetic activation involves the relaxation of the superstructure of chromatin, which may be, at least in part, modulated by its interaction with the nuclear matrix. Moreover, recent observations suggest that gene expression can be stimulated by alterations in the organization of the cytoskeleton. Therefore, we have characterized the changes in composition that the nuclear matrix-intermediate filament complex undergoes during the evolution of rat hepatocyte nodules. Dramatic changes in the expression of both the nuclear matrix and intermediate filament proteins occur during transformation; they are, however, related in a different way to the stages of carcinogenesis. Several new nuclear matrix proteins appear in early nodules, isolated 9 weeks after initiation. The subsequent evolution of persistent nodules is also characterized by discrete changes in the composition. Thus, the new synthesis of nuclear matrix proteins reflects the emergence of successive cellular populations, in line with the recent finding that a subset of components of the nuclear matrix is cell type-specific. In contrast, intermediate filament proteins undergo continuing changes. A new keratin with apparent molecular weight of 39 kDa, analogous to human keratin 19, appears in early nodules, and its expression steadily increases up to the 32nd week from initiation; at the same time, the amount of the proteolytic fragments of keratins A and D increases sharply. These findings suggest that the inappropriate expression of keratin 19 may be involved in the epigenetic activation of new cellular programs, through the rearrangement of the cytoskeleton which in turn may perturb nuclear matrix function.

Asunto(s)

Citoesqueleto/metabolismo , Neoplasias Hepáticas/patología , Proteínas Nucleares/metabolismo , Transformación Genética/fisiología , Animales , Antígenos Nucleares , Biomarcadores , Cationes Bivalentes/metabolismo , Cromosomas/fisiología , Citoesqueleto/química , Electroforesis en Gel Bidimensional , Endopeptidasas/metabolismo , Histonas/análisis , Humanos , Immunoblotting , Filamentos Intermedios/genética , Filamentos Intermedios/metabolismo , Isomerismo , Queratinas/biosíntesis , Queratinas/química , Neoplasias Hepáticas/química , Masculino , Peso Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Fenotipo , Ratas , Ratas Endogámicas F344

RESUMEN

Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism by which this protection is mediated is unclear. Since animal studies strongly suggest that production of endothelium-derived relaxing factor is enhanced by estradiol, we have examined the effect of estrogens on nitric oxide (NO) synthase (NOS) activity, protein, and mRNA in cultured bovine aortic endothelial cells. In reporter cells rich in guanylate cyclase, it has been observed that long-term treatment (> or = 24 hr) with ethinylestradiol (EE2) dose-dependently increased guanylate cyclase-activating factor activity in the conditioned medium of endothelial cells. However, conversion of L-[14C]arginine to L-[14C]citrulline by endothelial cell homogenate or quantification of nitrite and nitrate released by intact cells in the conditioned medium did not reveal any change in NOS activity induced by EE2 treatment. Similarly, Western and Northern blot analyses did not reveal any change in the endothelial NOS protein and mRNA content in response to EE2. However, EE2 dose- and time-dependently decreased superoxide anion production in the conditioned medium of endothelial cells with an EC50 value (0.1 nM) close to that which increased guanylate cyclase-activating factor activity (0.5 nM). Both of these effects were completely prevented by the antiestrogens tamoxifen and RU54876. Thus, endothelium exposure to estrogens appears to induce a receptor-mediated antioxidant effect that enhances the biological activity of endothelium-derived NO. These effects could account at least in part for the vascular protective properties of these hormones.

Asunto(s)

Endotelio Vascular/metabolismo , Etinilestradiol/farmacología , Expresión Génica/efectos de los fármacos , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico/metabolismo , Superóxidos/antagonistas & inhibidores , Animales , Aorta , Northern Blotting , Western Blotting , Bovinos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Cinética , ARN Mensajero/metabolismo , Factores de Tiempo

RESUMEN

In a series of related papers, we have recently presented the results of a thermodynamic approach to the conformational transitions of bulk chromatin induced in vitro by different structure-perturbing agents, such as the intercalating dye ethidium bromide or the ionic strength. In all these studies, we took advantage of the capability of differential scanning calorimetry to detect the changes in the melting behavior of the structural domains of chromatin (the linker and the core particle) associated with the order-disorder transitions. This technique also revealed that the higher-order structure undergoes a catastrophic decondensation process in the course of the transformation of rat hepatocytes as well as of cultured cells. Therefore, several questions arose concerning the biological function (if any) of the changes in the degree of condensation of bulk chromatin, as well as the mechanism of transition and the nature of the modulating agents. In this paper, we report a thermodynamic analysis of the reconstitution of H1-depleted calf thymus chromatin with the purpose of establishing (1) the binding mode of H1 and (2) the energetics and cooperativity of the transition from the unfolded to the condensed state. When H1 is progressively extracted from calf thymus nuclei by high-salt treatment, the endotherm at 107 degrees C, characteristic of the core particles interacting within condensed domains, converts into the thermal transition at 90 degrees C, resulting from the denaturation of noninteracting core particles. Binding of H1 fully restores the thermal profile of native chromatin. Analysis of H1 association shows that binding occurs at independent sites with KA = (3.67 +/- 0.60) x 10(4) M-1 and each site comprising 180 +/- 10 bp. The experimental dependence of the fraction of condensed chromatin on R, the moles of bound H1 per nucleosome mole, was compared with a simple thermodynamic model for the conformational change. This analysis yields a value of -5 kcal per nucleosome mole for the interaction free energy of nucleosomes within the ordered state. The process of condensation, is not, however, a highly cooperative (all-or-none) one, as expected from a consideration of the solenoidal model for the 30 nm fiber. Rather, nucleation of the helical state involves the face-to-face interaction between consecutive core particles, and the growth is largely determined by the mergence and rearrangement of neighboring clusters of helically arrayed nucleosomes.

Asunto(s)

Cromatina/química , Histonas/química , Pliegue de Proteína , Animales , Rastreo Diferencial de Calorimetría , Bovinos , Dicroismo Circular , Modelos Químicos , Unión Proteica , Conformación Proteica , Termodinámica

RESUMEN

Using differential scanning calorimetry and complementary ultrastructural observations, we have characterized the status of chromatin during the transformation of rat hepatocytes in the resistant hepatocyte model of Solt and Farber (1976. Nature (Lond.). 263:701-703). Differential scanning calorimetry affords a measure of the degree of condensation of chromatin in situ and has therefore been used in this work for the purpose of establishing the nature of the structural changes associated with the emergence of successive cellular populations. Since the resistant hepatocyte model generates a series of synchronous phenotypic changes, it was possible to determine unambiguously the content of heterochromatin at each step of the process. The higher-order structure undergoes a partial relaxation in early developing nodules, isolated 16 weeks after initiation; the thermal transition at 90 degrees C, which is characteristic of noninteracting core particles, increases with respect to control hepatocytes. Dramatic changes occur in persistent (46-week) nodules. The 90 degrees C endotherm dominates the thermogram, while the transition at 107 degrees C, corresponding to the denaturation of the core particle packaged within the heterochromatic domains, disappears. The complete loss of the higher-order structure at this stage of transformation has been further verified by ultrastructural observations on thin nuclear sections. Ten-nm filaments, having a beaded appearance, are scattered throughout the nucleoplasm and clearly result from the decondensation of 30-nm-thick fibers. This catastrophic relaxation process cannot be related to an effective increase in gene activity. Rather, our observations suggest that during transformation chromatin is in a state of high transcriptional competence associated with the alert of general cellular programs. This view is consistent with the finding that in persistent nodules the DNA is extensively hypomethylated with respect to normal liver.

Asunto(s)

Transformación Celular Neoplásica , Cromatina/química , Cromatina/ultraestructura , Neoplasias Hepáticas Experimentales/etiología , Animales , Fenómenos Biofísicos , Biofisica , Rastreo Diferencial de Calorimetría , Hígado/química , Hígado/ultraestructura , Neoplasias Hepáticas Experimentales/química , Neoplasias Hepáticas Experimentales/ultraestructura , Masculino , Microscopía Electrónica , Conformación de Ácido Nucleico , Ratas , Ratas Endogámicas F344 , Factores de Tiempo

RESUMEN

In cells latently infected with Epstein-Barr virus (EBV), the expression of two viral transactivators, EB1 and R, is responsible for the switch from latency to a productive cycle. R contains a DNA-binding/dimerization domain localized at the N-terminus. The domain required for transcriptional activation is localized at the C-terminus and contains two regions of very different amino acid composition. The first is very rich in prolines, whereas the second is rich in acidic residues and contains two potential alpha-helices. We investigated the activation potential of these subregions when linked to the heterologous Gal4 DNA-binding domain. We found that the acidic region--more precisely, the second putative alpha-helix--is an activating domain. In contrast, the proline-rich region is insufficient by itself for activation but collaborates with the acidic region in a cell-specific manner to make transactivation more efficient. We demonstrated that R interacts in vitro with the basal transcription factors TBP and TFIIB, and that the acidic domain of R mediates these interactions.

Asunto(s)

Herpesvirus Humano 4/metabolismo , Proteínas Inmediatas-Precoces , Transactivadores , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular , ADN Recombinante/genética , Herpesvirus Humano 4/genética , Humanos , Datos de Secuencia Molecular , Prolina/genética , Prolina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción TFIIB , Factor de Transcripción TFIID , Factores de Transcripción/genética , Proteínas Virales