Biomedical subjects

P Bushel

Publications and source records attributed to P Bushel.

10 recordsLinked to original sources

PubMed2002

Detection of diluted gene expression alterations using cDNA microarrays.

The use of DNA microarrays has spanned numerous disciplines of life science research. Despite the volume of studies utilizing this technology, no consensus exists on basic issues such as the determination of significantly altered genes in a given experiment, often leading to either false-negative or false-positive data. In this report, we study the effect of dilution of biological alterations on the detection level of gene expression differences using cDNA microarrays. We propose that subtle alterations in transcript levels of genes below the 2-fold level should be considered when replicate hybridizations are performed, because these subtle gene expression changes may be due to a robust response in few cells. We measured the effect of dilution of gene expression and found that differences in gene expression between the two cell lines assayed (HaCaT and MCF-7) were detected even after a 20-fold dilution factor. These results better our understanding of biological alterations that comprise a relatively small percentage of an assayed organ and help in the interpretation of gene expression data.

Cell Line↗

PubMed2001

Discovery in toxicology: mediation by gene expression array technology.

Toxicogenomics is a term that represents the merging of toxicology with novel genomics techniques. Data generated in the new-age era of toxicology is relatively complex, requires new bioinformatics tools for adequate interpretation, and allows for the rapid generation of testable hypotheses. Hazard identification and risk assessment processes will advance from the use of genomics techniques, which will lead to greater understanding of mechanism(s) of action of toxicants, development of novel biomarkers of exposure and effect, and better identification of sensitive subpopulations.

Animals↗

PubMed2001

Assessing gene significance from cDNA microarray expression data via mixed models.

The determination of a list of differentially expressed genes is a basic objective in many cDNA microarray experiments. We present a statistical approach that allows direct control over the percentage of false positives in such a list and, under certain reasonable assumptions, improves on existing methods with respect to the percentage of false negatives. The method accommodates a wide variety of experimental designs and can simultaneously assess significant differences between multiple types of biological samples. Two interconnected mixed linear models are central to the method and provide a flexible means to properly account for variability both across and within genes. The mixed model also provides a convenient framework for evaluating the statistical power of any particular experimental design and thus enables a researcher to a priori select an appropriate number of replicates. We also suggest some basic graphics for visualizing lists of significant genes. Analyses of published experiments studying human cancer and yeast cells illustrate the results.

Computational Biology↗

PubMed1995

Two serum response elements mediate transcriptional repression of human smooth muscle alpha-actin promoter in ras-transformed cells.

The mechanism by which activated ras oncogene expression leads to repression of genes encoding specific actin filament proteins is not understood. However, these changes associated with loss of organized actin filaments, are necessary to maintain the transformed phenotype. The human smooth muscle (sm) alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cell lines. In this study, we demonstrate that two serum response elements (SREs) present in the alpha-actin promoter are required for transcriptional repression in ras-transformed cells and the two SREs act synergistically to repress heterologous promoters in a ras-transformation dependent manner. Serum response factor (SRF), which can bind to the sm alpha-actin SREs, restores alpha-actin promoter activity in ras-transformed cells. c-Fos, c-Jun and YY1 also repress alpha-actin promoter through SREs, suggesting that these transcription factors may play a role in repressing alpha-actin promoter in ras-transformed cells.

Actins↗

PubMed1995

Activation of smooth muscle alpha-actin promoter in ras-transformed cells by treatments with antimitotic agents: correlation with stimulation of SRF:SRE mediated gene transcription.

Smooth muscle alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cells. We have recently shown that serum response factor (SRF) which can bind to the serum response elements (SREs) present in the alpha-actin promoter, can activate alpha-actin promoter activity in ras-transformed cells and suppress transformation by ras. Agents that stimulate SRF expression and alpha-actin promoter activity in ras-transformed cells are expected to be potential candidates as antitumor agents. In this study, we show that treatment of ras-transformed cells with antitumor agents such as taxol, vincristine, vinblastine, colchicine, and nocodazole leads to 5- to 7-fold activation of alpha-actin promoter driven CAT activity, whereas there was very little effect on thymidine kinase promoter driven CAT activity. This activation occurred at subcytotoxic concentrations of these agents and correlated with inhibition of cell cycle progression. Furthermore, these agents stimulated SRF expression in ras-transformed cells, as measured by its SRE binding activity. The increase in alpha-actin expression is accompanied by the restoration of actin filaments into organized bundles. These results suggest a novel mechanism by which antimitotic agents suppress the ras-transformed phenotype.

Actin Cytoskeleton↗

PubMed1995

Two proximal CArG elements regulate SM alpha-actin promoter, a genetic marker of activated phenotype of mesangial cells.

Mesangial cells express smooth muscle alpha-actin (SM alpha-actin) in response to glomerular injury in vivo, and SM alpha-actin gene expression serves as a genetic marker characterizing the activated phenotype of mesangial cells. We used a molecular genetic approach to analyze the SM alpha-actin promoter and evaluate transcriptional mechanisms that might direct the genetic switch of mesangial cells to the activated phenotype. The sequence spanning -894 to +1 of the SM alpha-actin promoter directed high levels of transcription that were attenuated in serum-restricted cells and upregulated upon treatment with serum or endothelin-1. Deletional analysis revealed a core promoter fragment, from positions -122 to +1, that was necessary and sufficient for transcription. This core activity was modulated by upstream sequences between -670 and -122. The 122-bp core promoter contains two highly conserved CArG box motifs (designated CB1 and CB2), and introduction of deletion mutations of either CB1 or CB2 reduced transcription in mesangial cells to near basal levels. Further analysis revealed that CB1 and CB2 acted synergistically when subcloned upstream of a heterologous, minimal thymidine kinase promoter. CB2 alone was sufficient to confer serum inducibility to a heterologous promoter, but both CB2 and CB1 were required for maximal levels of serum-induced transcription. Collectively, these results demonstrate that CB1 and CB2 cooperate to mediate serum-induced activation of the SM alpha-actin promoter in mesangial cells.

Actins↗

PubMed1992

Regulation of smooth muscle alpha-actin promoter in ras-transformed cells: usefulness for setting up reporter gene-based assay system for drug screening.

Oncogenic activation of ras results in changes in the transcription of several genes leading to uncontrolled cell growth. In this paper, we demonstrate that transformation of fibroblast cells by the ras oncogene leads to transcriptional repression of the smooth muscle alpha-actin promoter. Transient transfection analysis of plasmids containing the 5' upstream region of the human alpha-actin gene fused to human growth hormone or bacterial chloramphenicol acetyltransferase coding sequences into Rat-2 and ras-transformed Rat-2 (HO6) cells indicates that alpha-actin promoter is repressed in ras-transformed cells. In addition, stable rat fibroblast cell lines expressing human growth hormone or beta-galactosidase under the control of alpha-actin promoter exhibit repressed reporter gene activity following transformation by the ras oncogene. alpha-Actin promoter-driven beta-galactosidase activity is derepressed in revertants of ras-transformed stable cell lines. This revertant cell line expresses elevated levels of ras p21 protein and is resistant to retransformation by Ki and Ha-ras oncogenes. The revertant may have either a defective target protein whose activity is essential for the transforming activity of ras or an activated tumor suppressor gene which can suppress the activity of ras. These results indicate that smooth muscle alpha-actin promoter activity is a sensitive marker to follow phenotypic changes following transformation by ras and subsequent reversion. The advantages of this alpha-actin promoter-reporter gene assay system to screen for drugs that inhibit the transforming activity of ras, either directly or indirectly, are discussed.

Actins↗

PubMed1990

Correlation between patterns of DNase I-hypersensitive sites and upstream promoter activity of the human epsilon-globin gene at different stages of erythroid development.

DNA 5' to the human epsilon-globin gene exhibits unique patterns of DNase I-hypersensitive sites (DHS) in three human erythroleukemic cell lines which represent the embryonic (K562), fetal (HEL), and adult (KMOE) stages of erythroid development. We have mapped 10 epsilon-globin DHS in K562 cells, in which the epsilon-globin gene is maximally active. Major sites are located -11.7, -10.5, -6.5, -2.2 kilobase pairs (kbp) and -200 base pairs (bp) upstream of the gene and directly over the major cap site. Minor sites are located -5.5, -4.5, and -1.48 kbp and -900 bp upstream of the cap site. In HEL cells, in which the epsilon-globin gene is expressed at extremely low levels, the -11.7-, -10.5-, -5.5-, -4.5-, and -2.2-kbp DHS are no longer detectable; the -200-bp site is approximately 300-fold less sensitive to DNase I; and the -1.48-kbp, -900-bp, and major cap site DHS are 3- to 4-fold less sensitive. Only the DHS located -6.5 kbp relative to the major cap site is detectable at all three stages of erythroid development, including KMOE cells in which epsilon-globin synthesis is undetectable. We suggest that this site may be implicated in maintaining the entire beta-globin cluster in an active chromatin conformation. The five DHS downstream of the -6.5-kbp element possess associated promoters. Thus two distinct types of DHS exist--promoter positive and promoter negative. In HEL cells, all the upstream promoters are inactivated, although the -1.48-kbp and -900- and -200-bp DHS are still present. This suggests that the maintenance of DHS and regulation of their associated promoters occur by independent mechanisms. The inactivation of the upstream promoters in HEL cells while the major cap site remains active represents a unique pattern of expression and suggests that HEL cells possess regulatory factors which specifically down regulate the epsilon-globin upstream promoters.

Blotting, Southern↗

PubMed1990

Negative regulation of the human epsilon-globin gene by transcriptional interference: role of an Alu repetitive element.

The human epsilon-globin gene has a number of alternative transcription initiation sites which correspond with regions of DNase I hypersensitivity upstream of the canonical cap site. Transcripts originating from the promoters located -4.3/-4.5 and -1.48 kilobase pairs (kbp) and -900 and -200 base pairs (bp) upstream of the major epsilon-globin cap site can, at certain stages of erythroid differentiation, extend through the gene and are polyadenylated. The 350-bp PolIII transcripts, originating within the Alu repetitive element -2.2 kbp upstream of the cap site, extend in the opposite direction from the gene, are nonpolyadenylated, nucleus confined, and are detectable only in mature K562 cells or mature embryonic red blood cells where the epsilon-globin major cap site is maximally transcribed. Fragments containing the promoters located between -4.5 and -4.3 kbp upstream of the gene down regulate transcription from the epsilon-globin gene 20- to 30-fold in a transient expression assay in which both erythroid and nonerythroid cell lines were used. This occurs only when the direction of transcription from the -4.3/-4.5-kbp promoters is towards the gene, and we hypothesize that down regulation is caused by transcriptional interference. Fragments containing the Alu repetitive element -2.2 kbp upstream of the gene can overcome down regulation of the epsilon-globin gene by the -4.5-kbp element when interposed in the direct orientation between this element and the epsilon-globin gene.

Cell Line↗

PubMed1990

A short, highly repetitive element in intron -1 of the human c-Ha-ras gene acts as a block to transcriptional readthrough by a viral promoter.

We have identified a short, highly repetitive element within intron -1 of the human c-Ha-ras gene. This element was found to be transcribed in both orientations and to be homologous to heterogeneous nonpolyadenylated transcripts. The repetitive element blocked transcriptional readthrough from a strong upstream viral promoter but allowed unimpaired readthrough from the c-Has-ras promoter. We suggest that it may serve to prevent excessive transcription into the coding region of the gene under such circumstances as viral insertion.

Cell Line↗