Annexin A6 inhibits Ras signalling in breast cancer cells.

Overexpression of epidermal growth factor receptor (EGFR) is associated with enhanced activation of wild-type (hyperactive) Ras in breast cancer. Little is known about the regulation of Ras inactivation and GTPase-activating proteins (GAPs), such as p120GAP, in cells with hyperactive Ras. Recently, we showed that in EGFR-overexpressing A431 cells, which lack endogenous Annexin A6 (AnxA6), ectopic expression of AnxA6 stimulates membrane recruitment of p120GAP to modulate Ras signalling. We now demonstrate that, AnxA6 is downregulated in a number of EGFR-overexpressing and estrogen receptor (ER)-negative breast cancer cells. In these cells, AnxA6 overexpression promotes Ca(2+)- and EGF-inducible membrane targeting of p120GAP. In ER-negative MDA-MB-436 cells, overexpression of p120GAP, but not CAPRI or a p120GAP mutant lacking the AnxA6-binding domain inhibits Ras/MAPK activity. AnxA6 knockdown in MDA-MB-436 increases Ras activity and cell proliferation in anchorage-independent growth assays. Furthermore, AnxA6 co-immunoprecipitates with H-Ras in a Ca(2+)- and EGF-inducible manner and fluorescence resonance energy transfer (FRET) microscopy confirmed that AnxA6 is in close proximity of active (G12V), but not inactive (S17N) H-Ras. Thus, association of AnxA6 with H-Ras-containing protein complexes may contribute to regulate p120GAP/Ras assembly in EGFR-overexpressing and ER-negative breast cancer cells.

Analytical and biologic variability in measures of hemostasis, fibrinolysis, and inflammation: assessment and implications for epidemiology.

An increasing number of cardiovascular epidemiologic studies are measuring non-traditional risk markers of disease, most of which do not have established biovariability characteristics. When biovariability data have been reported, they usually represent a short time period, and, in any case, there is little consensus on how the information should be used. The authors performed a long-term (6-month) repeated measures study on 26 healthy individuals, and, using a nested analysis of variance (ANOVA) approach, report on the analytical (CVA), intraindividual (CVI), and between individual (CVG) variability of 12 procoagulant, fibrinolysis, and inflammation assays, including total cholesterol for comparison. The results suggest acceptable analytical variability (CVA < or = 1/2 CVI) for all assays. However, there was a large range of intraindividual variation as a proportion of total variance (2-78%), and adjusting for intraindividual and between individual variation in bivariate correlations increased the observed correlation by more than 30 percent for three of these assays. Overall, the assays showed a significant increase in intraindividual variation over 6 months (p < 0.05). While these findings suggest that most of these assays have biovariability characteristics similar to cholesterol, there is variation among assays. Some assays may be better suited to epidemiologic studies, and knowledge of an assay's biovariability data may be useful in interpreting simple statistics, and in designing multivariate models.

The preoperative bleeding time test lacks clinical benefit: College of American Pathologists' and American Society of Clinical Pathologists' position article.

The major conclusions of this position article are as follows: (1) In the absence of a history of a bleeding disorder, the bleeding time is not a useful predictor of the risk of hemorrhage associated with surgical procedures. (2) A normal bleeding time does not exclude the possibility of excessive hemorrhage associated with invasive procedures. (3) The bleeding time cannot be used to reliably identify patients who may have recently ingested aspirin or nonsteroidal anti-inflammatory agents or those who have a platelet defect attributable to these drugs. The best preoperative screen to predict bleeding continues to be a carefully conducted clinical history that includes family and previous dental, obstetric, surgical, traumatic injury, transfusion, and drug histories. A history suggesting a possible bleeding disorder may require further evaluation; such an evaluation may include performance of the bleeding time test, as well as a determination of the platelet count, the prothrombin time, and the activated partial thromboplastin time. In the absence of a history of excessive bleeding, the bleeding time fails as a screening test and is, therefore, not indicated as a routine preoperative test.

Id genes encoding inhibitors of transcription are expressed during in vitro astrocyte differentiation and in cell lines derived from astrocytic tumors.

Id proteins belong to a class of nuclear transcription factors known as helix-loop-helix proteins. It has been reported that Id genes function as negative regulators of differentiation, and Id gene expression is down-regulated during cell differentiation. We examined the regulation of Id genes during astrocyte differentiation in a murine nervous system precursor cell line, NSEHip2-28, which is able to differentiate along the astroglial lineage, as well as in human astroglial tumor cell lines. Upon induction of NSEHip2-28 differentiation, at a time when glial fibrillary acidic protein expression became detectable, the expression of all four Id family members initially increased dramatically, and subsequently decreased. Furthermore, varying levels of Id gene expression were found in astroglial tumor cell lines displaying variable degrees of lineage-specific differentiation. These results suggest that the expression of Id family members may play an important role in the control of astrocyte differentiation.

Variability in the measurement of C-reactive protein in healthy subjects: implications for reference intervals and epidemiological applications.

We developed a reproducible ELISA for C-reactive protein (CRP), calibrated with WHO Reference Material, for which intra- and interassay CVs were 3.0% and 6.0%, respectively. Analytical recovery was 97.9%. The distribution of CRP in a healthy blood donor population (n = 143) was nongaussian, with 2.5th, 50th, and 97.5th percentile values of 0.08, 0.64, and 3.11 mg/L, respectively. There was no sex-related difference, and the association with age was weak. In a study of variability [by the method of Fraser and Harris (Crit Rev Clin Lab Sci 1989;27:409-37)], the analytical variability was 5.2%; the within-subject variability, CVI, was 42.2%; and the between-subject variability, CVG, was 92.5%. The critical difference for sequential values significant at P < or =0.05 (i.e., the smallest percentage change unlikely to be due to analytical variability or CVI) was calculated as 118%, and the index of individuality, CVI/CVG, was 0.46. This suggests that CRP, like many clinical chemistry analytes, has limited usefulness in detecting early disease-associated changes when used in conjunction with a healthy reference interval. From a molecular epidemiological standpoint, the usefulness of CRP in longitudinal studies is suggested by the small index of individuality and by observations that (a) short-term fluctuations were infrequent, (b) all data stayed within the reference interval, and (c) relative rankings of the subjects over 6 months only moderately deteriorated.

Evidence that meizothrombin is an intermediate product in the clotting of whole blood.

Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospholipid surface. The biological significance of this intermediate has recently been challenged by the apparent absence of meizothrombin during clotting of sodium citrate-anticoagulated plasma. We analyzed the formation of thrombin during coagulation of nonanticoagulated, unchilled, minimally manipulated whole blood in glass tubes. The process was stopped at 0, 3, 5, and 7 minutes by the addition of biotinylated peptidyl chloromethyl-ketone active-site labeling reagents. Plasma/serum was separated by centrifugation, and labeled species were extracted by immunoadsorption with a polyclonal anti-prothrombin antibody. The purified prothrombin-derived species were separated by SDS-polyacrylamide gradient gel electrophoresis and visualized on a chemiluminescent avidin blot. Meizothrombin appeared as an intermediate product of this reaction and persisted with some increase through the 7-minute time point. We also observed incorporation of the active-site label into a species of lower molecular weight consistent with the B1 chain of beta- and/or gamma-thrombin. These degraded forms of thrombin have not been previously demonstrated in a biologically relevant preparation. Our data clearly establish the generation of meizothrombin as an intermediate product of thrombin generation during whole-blood clotting. The data also represent the first experimental evidence for the generation of beta- and gamma-thrombin in a biologically relevant environment and time scale.

Factor VII assays.

Several prospective and cross-sectional studies have proposed that an association exists between elevated coagulation factor VII levels and cardiovascular disease. Not all of these studies used the same method to assess the factor VII levels. Although the most common method is the one-stage factor assay, there are numerous variables in the composition of this assay. Also, factor VII may circulate in plasma in several forms. The relative contribution of each of these forms to the assay result is presently unknown. Future efforts may help to standardize factor VII assays, improve understanding of the influence of the various forms of factor VII, and identify nonfactor VII components that may both affect assay results and be potential indicators for risk of cardiovascular disease.

Performance characteristics of fibrinogen assays. Results of the College of American Pathologists Proficiency Testing Program 1988-1991.

Plasma fibrinogen concentration appears to be an important risk factor for the development of atherosclerotic cardiovascular disease of a similar magnitude to cholesterol. The quality control of plasma fibrinogen assays has taken on new importance as a consequence of this potential role as an atherosclerotic risk factor. This article reviews the performance characteristics of 40,000 fibrinogen assays comprising the College of American Pathologists Proficiency Testing Program from 1988 through 1991. Instrument and reagent variables both play roles in the poor interlaboratory reproducibility documented by this study. The absence of either a national or international standard for plasma fibrinogen assays has been a major source of reagent variability. The validation and calibration of the College of American Pathologists lyophilized plasma reference preparation for fibrinogen determination is also reported in this study. The availability of this validated reference plasma should markedly improve interlaboratory reproducibility.

An embryonic origin for medulloblastoma.

Medulloblastoma is a common brain tumor of children. Three differentiated cell types are found in medulloblastomas: neurons, glia, and muscle cells. Because of the presence of multiple differentiated cell types these tumors were named after a postulated cerebellar stem cell, the medulloblast, that would give rise to the differentiated cells found in the tumors. We describe a cell line with the properties expected of the postulated medulloblast. The rat cerebellar cell line ST15A expresses an intermediate filament, nestin, that is characteristic of neuroepithelial stem cells. ST15A cells can differentiate, gaining either neuronal or glial properties. In this paper we show that the same clonal cell can also differentiate into muscle cells. This result suggests that a single neuroectodermal cell can give rise to the different cell types found in medulloblastoma. We also show expression of nestin in human medulloblastoma tissue and in a medulloblastoma-derived cell line. Both the properties of the ST15A cell line and the expression of nestin in medulloblastoma support a neuroectodermal stem cell origin for this childhood tumor.

Downregulation of CDC2 upon terminal differentiation of neurons.

The terminal differentiation of neurons occurs as precisely timed waves, with specific neuronal types differentiating in defined sequences. The precision of neuronal differentiation in the central nervous system offers an unusual opportunity to study terminal differentiation in vivo. The p34cdc2 kinase complex and the anti-oncogenes p53 and RB are central in the regulatory network that controls cell proliferation. We found high levels of expression of CDC2 mRNA and protein in proliferating neuronal precursor cells. The expression of both CDC2 and cyclin A was dramatically downregulated upon terminal differentiation of neurons in vivo and in a neuronal precursor cell line, ST15A. p53 mRNA expression was also downregulated but to a lesser extent; RB mRNA levels were unchanged during neuronal differentiation. Immunohistochemistry showed that p34cdc2 was expressed not only in the neuronal precursors of the cerebellar external granule layer but also in the early differentiating granule neurons. The expression of p34cdc2 in early neurons suggests a function for this enzyme in the events that occur soon after proliferation ceases. On the basis of the results reported here and other recent findings, we propose a model in which terminal differentiation is achieved by a switch in the neuronal precursors from p34cdc2-based proliferation to a differentiated state controlled by p34cdc2-related kinases.

The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter.

The c-fos proto-oncogene is rapidly and transiently induced by a variety of extracellular stimuli. We have previously shown that conditioned media from v-sis transformed NRK cells rapidly induces a DNA binding protein which binds to a conserved sequence upstream of the human c-fos gene. We now show that purified recombinant c-sis/PDGF can induce this binding activity which we have termed SIF, for sis-inducible factor. Oligonucleotides which bind to the SIF protein will confer sis/PDGF inducibility onto a truncated, unresponsive c-fos promoter. However, sequences lying between -100 and -57 of the c-fos gene are required for this induction. The sis-responsive element functions independently of a region of dyad symmetry previously identified as the serum responsive element (SRE). The time course of c-fos expression driven by the sis-responsive element is similar to that mediated by the SRE. Unlike the SRE, which can respond to signals generated by sis/PDGF, serum or phorbol esters, the SIF binding element mediates c-fos induction only in response to sis/PDGF. The SRE and SIF elements function in an additive manner to stimulate the transcription of the c-fos gene in response to sis/PDGF.

The human c-fos serum response factor and the yeast factors GRM/PRTF have related DNA-binding specificities.

One of the elements that mediates growth factor and serum inducibility of the human c-fos gene is a region of dyad symmetry that lies between nucleotides -320 and -299 of the human gene. A mammalian protein specifically binds to this sequence element and has been termed the serum response factor (SRF). Gel-shift analysis and competition experiments demonstrate that there is a factor in the yeast Saccharomyces cerevisiae that binds specifically to the human c-fos SRE. The methylation interference pattern of the yeast factor is identical to that of the mammalian SRF. Regulatory elements of cell-type-specific genes in yeast have homologies to the c-fos SRE and complete for binding of both the mammalian and yeast factors to the SRE. Antisera to the gene product of the MCM1 locus react with the yeast SRE-binding factor. These data suggest that this yeast protein is closely related or identical to the factors [general regulator of mating type (GRM) and pheromone/receptor transcription factor (PRTF)] that are required for the regulation of cell-type-specific genes in yeast.

Examination of cerebrospinal fluid in the horse.

The examination of cerebrospinal fluid (CSF) is often part of the diagnostic work-up of a patient exhibiting signs of disease involving the central nervous system. Awareness of the capabilities and limitations of these laboratory tests is important in assessing the benefit-to-risk ratio of performing such procedures. Collection of CSF is a relatively simple procedure, and together with a thorough history, physical examination, and other diagnostic tests, may be a valuable aid in arriving at a diagnosis or prognosis.

Identification of the promoter sequences involved in the cell specific expression of the rat somatostatin gene.

DNA sequences containing the 5' flanking region of the rat somatostatin gene were linked to the coding sequence of the bacterial chloramphenicol acetyl transferase gene. This recombinant plasmid is active in expressing CAT activity in the neuronally derived, somatostatin producing CA-77 cell line. Deletion analyses of the somatostatin promoter show that the sequences proximal to position -60, relative to the cap site are required for expression of this promoter. A 4 base pair deletion of residues -46 through -43 within the somatostatin promoter results in a down mutation in vivo suggesting the existence of an element critical for the expression of the promoter in CA-77 cells. In addition, the somatostatin recombinant and its 5' deletion constructs preferentially express CAT activity in CA-77 cells, whereas only basal level of expression is observed in HeLa, BSC40, and RIN-5F cell lines, pointing to the cell specific nature of this promoter.

Inducible binding of a factor to the c-fos regulatory region.

The c-fos gene is rapidly and transiently activated in quiescent BALB/c-3T3 cells in response to serum, platelet-derived growth factor or conditioned medium from v-sis-transformed cells. This activation occurs at the level of transcription and in the absence of new protein synthesis. Using a gel electrophoresis DNA-binding assay, we have found a DNA-binding activity in BALB/c-3T3 cells that is induced within 20 min of treatment with conditioned medium from v-sis-transformed cells. A DNA methylation interference assay has shown that this factor binds to a sequence approximately 346 base pairs upstream of the transcription initiation site of the human c-fos gene. Insulin, epidermal growth factor, and phorbol 12-myristate 13-acetate fail to induce this DNA-binding factor. Protein synthesis inhibitors do not block the induction of this activity. We propose that this factor preexists in an inactive form in quiescent cells and that its binding activity is activated in response to appropriate extracellular inducers.

A rapidly inducible DNA-binding activity which binds upstream of the c-fos proto-oncogene.

The c-fos proto-oncogene is rapidly inducible by a variety of extracellular stimuli. In order to dissect the intracellular signalling pathways responsible for c-fos induction, we have used a gel electrophoresis DNA-binding assay to identify trans-acting factors that bind to c-fos regulatory regions. We have identified a factor in Balb/c-3T3 cells that binds to an oligonucleotide, containing sequences -351 to -337 of the human c-fos gene. This factor is inducible in quiescent cells within 30 min of addition of conditioned medium from v-sis transformed cells. Cycloheximide fails to block this induction. This result suggests that the factor is present in an inactive form in quiescent cells and is activated only in response to the appropriate inducer. Fibroblast growth factor (FGF) and the tumor promoter phorbol myristate acetate (TPA) induce the c-fos gene but not this DNA-binding activity. We propose from this that there are multiple regulatory regions upstream of c-fos each capable of responding to a different set of stimuli.

Z-DNA in the rat somatostatin gene.

Two alternating purine-pyrimidine tracts flank the rat gene encoding the polypeptide hormone somatostatin. One lies 5' and one 3' to the gene; both consist of tandem repeats of the dinucleotide TG, with 25 repeats in the tract 5' and 15 in the tract 3' to the gene, and both are bordered on at least one edge by short repeated sequences. Characterization of supercoiled plasmids containing these sequences reveals that both form Z-DNA. Using S1 nuclease as a probe of DNA conformation we have investigated the fine structure of the Z-DNA and have shown: 1) that the entire Z-DNA segment as well as the single-stranded junctions flanking it is sensitive to S1 nuclease; 2) that the B-DNA/Z-DNA junction can be contained within the ends of the alternating purine-pyrimidine tract; and 3) that the sequences bordering the alternating purine-pyrimidine tracts affect the extent of Z-DNA propagation, sometimes as a result of their own apparently nonB-DNA conformation. We have also examined the published sequence of the human somatostatin gene (Shen, L.-P., and Rutter, W.J. (1984) Science 224, 168-171) for alternating purine-pyrimidine or potential Z-DNA-forming sequences and compared them to those present in the rat gene. We find that the human gene contains a 32-base pair alternating purine-pyrimidine sequence in an analogous position to the (TG)25 tract 5' to the rat gene, although the two sequences are not homologous. There are also six shorter alternating purine-pyrimidine elements 5' to the transcribed sequences which are positioned almost identically in the two genes with respect to the transcription initiation sites, although their sequences are not well conserved. We propose that the parallel placement of alternating purine-pyrimidine or potential Z-DNA-forming sequences 5' to the somatostatin genes from two species is a result of structural, in contrast to sequence, conservation. These observations suggest that the rat somatostatin gene may be a good model system for the investigation of the function of Z-DNA in the regulation of gene expression.

Isolation, characterization, and DNA sequence of the rat somatostatin gene.

The gene encoding rat somatostatin has been isolated from a lambda phage gene library. Phage harboring the gene were identified by plaque hybridization using a nick-translated fragment derived from the cDNA for rat somatostatin. The transcriptional unit includes exons of 238 and 367 base pairs (bp) separated by one intron of 621 bp. The intron is located between the codons for Gln (-57) and Glu (-56) of prosomatostatin. Analysis of the nucleotide sequence 5' to the start of transcription reveals a number of sequences which may be involved in the expression of somatostatin. A variant of the "TATA" box, TTTAAA, lies 26 bp upstream from the start of transcription, and a sequence homologous to the "CAAT" box (GGCTAAT) is 92 bp upstream from the transcription start. A long alternating purine-pyrimidine stretch, (GT)25, which is similar to Z DNA-forming sequences in other genes, lies 628 bp 5' to the transcription start and is flanked by small repeats. Hybridization analysis shows that this region is highly repeated in the genome and that homologous sequences are located approximately 2 kilobase pairs downstream from the poly(A) addition site. Southern hybridization of the lambda clone with probes derived from brain or liver poly(A+) RNA demonstrates that another transcribed sequence lies about 7 kilobase pairs downstream from the poly(A) addition site of the rat somatostatin gene. Analysis of rat DNA suggests that there may be restriction-site polymorphisms in or near the gene or that additional somatostatin-hybridizing sequences may exist in the genome.