Cross-sectional area of the median nerve after intraneural vs perineural low volume administration. / Evaluación del área de sección del nervio mediano tras la inyección intraneural o perineural.

INTRODUCTION: To recognise the relationship between the needle tip and the median nerve during peripheral nerve block is of interest to avoid neural damage. However, signs of intraneural injection are not clearly established. The aim of this study was to define the changes observed in the peripheral nerve after the intraneural or perineural administration of 1ml of solution. MATERIAL AND METHODS: Ultrasound guided median nerve blocks were performed in the forearm of 10 fresh cadavers on 60 occasions (3 per forearm). They were randomised into the intraneural (n=30) or perineural (n=30) location of the needle tip, after the consensus of location by 7 specialists. After 1ml of solution was injected an evaluation was made of the changes in the cross-sectional area of the nerve, as well as the displacement along the nerve. RESULTS: The cross-sectional area of the median nerve was increased in both groups, however, the increase was significantly higher in the intraneural group (perineural 0.007±0.013cm2 vs. intraneural 0.032±0.021cm2, P<.0001). An increase of more than 27% of the area ensures an intraneural injection in the median nerve according to the ROC curve analysis. Both proximal and distal diffusion were observed more frequently in the intraneural group (proximal: 86% vs 14%, P<.0001, Distal: 43% vs 4%, P<.0001). CONCLUSIONS: Based on this experimental study, it is concluded that the injection of a small volume (1ml) allows to discriminate the disposition of the intraneural vs perineural needle in a high percentage of cases. Therefore, it is suggested that this "dose test" should be considered in the safety algorithms if it is required to reduce the incidence of intraneural injection.

Rb targets histone H3 methylation and HP1 to promoters.

In eukaryotic cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1 (ref. 2), creating a binding site for the chromo domain of HP1 (refs 3, 4). Here we show that SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter, and in fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins.

Forkhead homologue in rhabdomyosarcoma functions as a bifunctional nuclear receptor-interacting protein with both coactivator and corepressor functions.

In a search for novel transcriptional intermediary factors for the estrogen receptor (ER), we used the ligand-binding domain and hinge region of ER as bait in a yeast two-hybrid screen of a cDNA library derived from tamoxifen-resistant MCF-7 human breast tumors from an in vivo athymic nude mouse model. Here we report the isolation and characterization of the forkhead homologue in rhabdomyosarcoma (FKHR), a recently described member of the hepatocyte nuclear factor 3/forkhead homeotic gene family, as a nuclear hormone receptor (NR) intermediary protein. FKHR interacts with both steroid and nonsteroid NRs, although the effect of ligand on this interaction varies by receptor type. The interaction of FKHR with ER is enhanced by estrogen, whereas its interaction with thyroid hormone receptor and retinoic acid receptor is ligand-independent. In addition, FKHR differentially regulates the transactivation mediated by different NRs. Transient transfection of FKHR into mammalian cells dramatically represses transcription mediated by the ER, glucocorticoid receptor, and progesterone receptor. In contrast, FKHR stimulates rather than represses retinoic acid receptor- and thyroid hormone receptor-mediated transactivation. Most intriguingly, overexpression of FKHR dramatically inhibits the proliferation of ER-dependent MCF-7 breast cancer cells. Therefore, FKHR represents a bifunctional NR intermediary protein that can act as either a coactivator or corepressor, depending on the receptor type.

The role of p21 in interferon gamma-mediated growth inhibition of human breast cancer cells.

IFN-gamma-mediated growth inhibition requires signal transducers and activators of transcription (STAT)-1 activation and may require induction of the cyclin-dependent kinase inhibitor p21. Using an electrophoretic mobility shift assay, we identified STAT1 activation after IFN-gamma treatment in breast cancer cell lines. Accordingly, IFN-gamma inhibited proliferation of monolayer cultured MCF-7 and MDA-MB-231 breast cancer cells. Interestingly, IFN-gamma inhibited anchorage-independent growth of MCF-7 cells but had no effect on MDA-MB-231 colony formation. Because p21 has been shown to play a role in anchorage-independent growth and is a transcriptional target of STAT1, we examined the effect of IFN-gamma on p21 mRNA. We found that IFN-gamma induced p21 mRNA in MCF-7 cells but not in MDA-MB-231 cells. Furthermore, IFN-gamma induced activation of a p21 promoter-luciferase reporter construct that contained the STAT1-inducible element in MCF-7 cells, but not in MDA-MB-231 cells. IFN-gamma treatment resulted in increased p21 protein in MCF-7 cells, whereas MDA-MB-231 cells did not appear to express detectable p21, even after IFN-gamma treatment. However, in MDA-MB-231 cells, p21 protein was detected only after proteosome inhibition, suggesting that degradation may be responsible for the undetectable level of p21 in these cells, despite the abundant mRNA levels. Finally, focus formation of MDA-MB-231 cells was inhibited by overexpression of p21. In conclusion, STAT1 activation does not appear to be sufficient for IFN-gamma-mediated growth inhibition. Furthermore, the role of p21 appears to be complex because monolayer growth inhibition occurs in the absence of p21, but anchorage-independent growth inhibition may require p21. Breast cancer cells may provide a unique model for further study of IFN-gamma signaling.

Chromatin structure analysis of the human c-fos promoter reveals a centrally positioned nucleosome.

Chromatin architecture plays an important regulatory role in nuclear transcription in eukaryotes. In particular, a preset chromatin organization has been proposed to prime rapidly inducible genes for response to extracellular signals. We analyzed the chromatin architecture of the rapidly inducible human c-fos gene using a combination of nuclease digestion studies. Several regions of nuclease sensitivity in the c-fos gene are observed: (i) a hypersensitive site at position -1900; (ii) a region centered at -350 that corresponds to the locations of the serum response element (SRE) and the sis-inducible element (SIE); (iii) a region around the transcriptional start site that includes the TATA box, the cAMP-responsive element (CRE) and a directly repeated sequence (DRE); and (iv) two sites at positions +250 and +550 that appear to delineate the linker regions of two positioned nucleosomes. In contrast, the region from -280 to -90 is strongly resistant to nuclease digestion. We identify a highly positioned nucleosome in this region of the c-fos promoter that may contribute to a defined higher-order nucleoprotein structure containing the nucleosome and proteins bound to the SIE, SRE, DRE, and CRE elements and TATA box. This structure may lead to a functional juxtaposition of the regulatory elements of the c-fos promoter and suggests that such a chromatin architecture is particularly suited for presetting promoters for rapid responsiveness.

Increased histone H1 phosphorylation and relaxed chromatin structure in Rb-deficient fibroblasts.

Fibroblasts derived from embryos homozygous for a disruption of the retinoblastoma gene (Rb) exhibit a shorter G1 than their wild-type counterparts, apparently due to highly elevated levels of cyclin E protein and deregulated cyclin-dependent kinase 2 (CDK2) activity. Here we demonstrate that the Rb-/- fibroblasts display higher levels of phosphorylated H1 throughout G1 with the maximum being 10-fold higher than that of the Rb+/+ fibroblasts. This profile of intracellular H1 phosphorylation corresponds with deregulated CDK2 activity observed in in vitro assays, suggesting that CDK2 may be directly responsible for the in vivo phosphorylation of H1. H1 phosphorylation has been proposed to lead to a relaxation of chromatin structure due to a decreased affinity of this protein for chromatin after phosphorylation. In accord with this, chromatin from the Rb-/- cells is more susceptible to micrococcal nuclease digestion than that from Rb+/+ fibroblasts. Increased H1 phosphorylation and relaxed chromatin structure have also been observed in cells expressing several oncogenes, suggesting a common mechanism in oncogene and tumor suppressor gene function.

TGF beta-induced growth inhibition in primary fibroblasts requires the retinoblastoma protein.

Transforming growth factor beta (TGF beta) inhibits cell proliferation by inducing a G1 cell-cycle arrest. Cyclin/CDK complexes have been implicated in this arrest, because TGF beta treatment leads to inhibition of cyclin/CDK activity. We have investigated the role of the retinoblastoma protein (pRb) in TGF beta-induced growth arrest by using RB+/+ and RB-/- primary mouse embryo fibroblasts. In both of these cell types, TGF beta inhibits CDK4-associated kinase activity. However, whereas CDK2-associated kinase activity was completely inhibited by TGF beta in the wild-type cells, it was reduced only slightly in the RB mutant cells. In addition, at high-cell density the growth-inhibitory effects of TGF beta are no longer observed in the RB-/- cells; on the contrary, TGF beta treatment promotes the growth of these mutant fibroblasts. Thus, under certain cellular growth conditions, elimination of pRb transforms the growth-inhibitory effects of TGF beta into growth-stimulatory effects. These observations could help to explain why TGF beta is often found to enhance tumorigenicity in vivo and why inactivation of the RB gene leads to tumorigenesis.

Altered cell cycle kinetics, gene expression, and G1 restriction point regulation in Rb-deficient fibroblasts.

Fibroblasts prepared from retinoblastoma (Rb) gene-negative mouse embryos exhibit a shorter G1 phase of the growth cycle and smaller size than wild-type cells. In addition, the mutant cells are no longer inhibited by low levels of cycloheximide at any point in G1 but do remain sensitive to serum withdrawal until late in G1. Certain cell cycle-regulated genes showed no temporal or quantitative differences in expression. In contrast, cyclin E expression in Rb-deficient cells is deregulated in two ways. Cyclin E mRNA is generally derepressed in mutant cells and reaches peak levels about 6 h earlier in G1 than in wild-type cells. Moreover, cyclin E protein levels are higher in the Rb-/- cells than would be predicted from the levels of its mRNA. Thus, the selective growth advantage conferred by Rb gene deletion during tumorigenesis may be explained in part by changes in the regulation of cyclin E. In addition, the mechanisms defining the restriction point of late G1 may consist of at least two molecular events, one cycloheximide sensitive and pRb dependent and the other serum sensitive and pRb independent.

Growth suppression by p16ink4 requires functional retinoblastoma protein.

p16ink4 has been implicated as a tumor suppressor that is lost from a variety of human tumors and human cell lines. p16ink4 specifically binds and inhibits the cyclin-dependent kinases 4 and 6. In vitro, these kinases can phosphorylate the product of the retinoblastoma tumor suppressor gene. Thus, p16ink4 could exert its function as tumor suppressor through inhibition of phosphorylation and functional inactivation of the retinoblastoma protein. Here we show that overexpression of p16ink4 in certain cell types will lead to an arrest in the G1 phase of the cell cycle. In addition, we show that p16ink4 can only suppress the growth of human cells that contain functional pRB. Moreover, we have compared the effect of p16ink4 expression on embryo fibroblasts from wild-type and RB homozygous mutant mice. Wild-type embryo fibroblasts are inhibited by p16ink4, whereas the RB nullizygous fibroblasts are not. These data not only show that the presence of pRB is crucial for growth suppression by p16ink4 but also indicate that the pRB is the critical target acted upon by cyclin D-dependent kinases in the G1 phase of the cell cycle.

Multiple basal promoter elements determine the level of human c-fos transcription.

Three cis-acting domains that contribute to the basal promoter activity of the human c-fos gene were identified. One encompasses the serum response element and has been previously described. Another spans an NF1-like site situated at -170. Mutations and in vitro protein binding assays pinpoint this site as the sole basal element of the medial domain. The third, or promoter-proximal, domain can be divided into several distinct sites, one containing a directly repeated GC-rich element and the other consisting of partially overlapping recognition sites for transcription factors ATF/CREB and MLTF/USF. Each of these sites contributes to basal activity as assayed by transient transfections and by in vitro transcription. Consistent with this, several complexes could be visualized between this region and nuclear proteins in vitro and genomic footprinting demonstrated that both elements are constitutively bound in vivo. On the basis of these results, we conclude that all three domains are necessary for full c-fos promoter function.

Rapid induction of c-fos transcription reveals quantitative linkage of RNA polymerase II and DNA topoisomerase I enzyme activities.

The functional association between DNA topoisomerase I and gene activity has been analyzed using the tightly regulated c-fos proto-oncogene, which undergoes rapid transitions between active and inactive states of transcription. We show that the topoisomerase I inhibitor camptothecin can be used to measure topoisomerase I activity throughout the transcription cycle of the c-fos gene. Upon induction of c-fos transcription in the presence of camptothecin, topoisomerase I cleavages spread through the gene in the 5' to 3' direction and concomitantly transcriptional elongation is retarded. Parallel kinetic measurements of RNA polymerase II activity and topoisomerase I activity demonstrate a quantitative and temporal link between the two enzymes. Our results argue that topoisomerase I quantitatively relieves the torsional consequences of transcriptional elongation in intact cells.

Occupation of the c-fos serum response element in vivo by a multi-protein complex is unaltered by growth factor induction.

Rapid, transient induction of the human c-fos proto-oncogene by extracellular signals requires the presence in cis of the serum response element (SRE). Two protein factors that bind to the SRE in vitro are the serum response factor (p67SRF) and polypeptide p62. These polypeptides must interact with one another and the SRE for efficient serum induction of the c-fos gene. Here we use dimethyl sulphate genomic footprinting to establish the in vivo protein contacts on the SRE and flanking sequences. In human A431 cells the patterns of protection and hyper-reactivity that we find are consistent with the presence of p67SRF, p62, and at least one other protein immediately 3' to p67SRF. The protein-DNA contacts we observe within the SRE are present before induction by epidermal growth factor and are unchanged during gene activation and subsequent repression. Our results indicate that a specific DNA-protein architecture may be maintained at the c-fos SRE, regardless of changes in the transcriptional state of the gene. Such established structures could be important generally in rapid transcriptional responses to extracellular signals.

Binding of anti-Z-DNA antibodies to negatively supercoiled SV40 DNA.

The binding of anti-Z-DNA antibody preparations to negatively supercoiled, protein-free SC40 DNA was analyzed. Covalent cross-linking with 0.1% glutaraldehyde followed by DNA restriction endonucleolytic fragmentation and nitrocellulose filtration allowed accurate mapping of antibody binding sites. The critical superhelical density necessary to allow antibody binding was -sigma = 0.056. The major region of antibody-DNA interaction was found within an SV40 segment spanning viral map positions 40 to 474. This region coincides with the nucleosome free region in SV40 minichromosomes and harbours the early and late promoter regions including the SV40 enhancer segment. Although it is unknown whether alternative, non-B-DNA conformations are generated in vivo within SV40 minichromosomes our results emphasize the high degree of DNA structural flexibility that can be realized under negative torsional stress.

DNA topoisomerase II cleaves at specific sites in the 5' flanking region of c-fos proto-oncogenes in vitro.

We have analyzed 1 kb of cloned human c-fos sequence (-711 to +287) for calf thymus DNA topoisomerase II cleavage sites in vitro. Using the anti-tumor drug VP16 (demethylepipodophyllotoxin-beta-D-glucoside) with purified topoisomerase II, we identify twelve sites. Five sites are clustered around position -306 in a region that possesses enhancer-like properties. A second cluster of three sites is positioned 15 bp upstream of the TATA promoter element. With a HeLa nuclear extract as a source of topoisomerase II, a subset of cleavage sites is conserved within the two clusters. The cleavage sites in the enhancer-like element are conserved in the homologous region of the murine c-fos. These findings raise the possibility that topoisomerase II is involved in mediation of mitogen-induced c-fos expression.