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At least 235 records · Page 13Linked to original sources
PubMed1986

Selective gene amplification in mammalian cells after exposure to 60Co gamma rays, 241Am alpha particles, or uv light.

Simian Virus 40 wild type (SV40)-transformed Chinese hamster embryo cells (Co631) contain about five viral copies integrated per cell genome. These SV40 sequences were used as endogenous indicator genes to study the response of mammalian cells to radiation at the gene level. An increase in copy number was detected by dispersed cell blotting and Southern analysis in combination with specific DNA hybridization. All types of radiation tested induce a 15- to 25-fold amplification of SV40 sequences without producing intact virus. The amplification is dose dependent and increases with time after irradiation: a maximum effect is observed at Day 3 after alpha particle or uv exposure and at Day 6 after gamma-ray exposure. A RBE of 6 can be calculated for alpha particles if amplification rates at Day 3 are compared. However, when the maximum effect is considered independent of time, no difference between different types of radiation is observed. Southern blots of genomic DNA show that not all integrated SV40 sequences are amplified upon radiation. Amplified sequences are found either in restriction fragments of relatively high molecular weight or in unit size fragments. SV40 amplification is selective in that the amplification of other genes, e.g., of alpha-actin, dhfr (dihydrofolate reductase), and of two oncogenes of the ras family (Kirsten ras and Harvey ras), was below detection level.

Alpha Particles↗
PubMed1984

Efficient DNA-mediated transfer of selectable genes and unselected sequences into differentiated and undifferentiated mouse melanoma clones.

We have found that three phenotypically dissimilar mouse B16 melanoma subclones are competent recipients for DNA-mediated gene transfer. Two of these approach and a third, amelanotic clone B78H1, surpasses mouse LTK cells in frequencies of transferent colony formation after treatment with either of two codominantly selectable plasmid vectors, pSV2gpt or pGCcos3neo. Melanoma transferents incorporate both selectable plasmid-homologous sequences and substantial amounts of unselected donor DNA into their cellular DNAs. In addition they retain the distinctive states of differentiation characteristic of the untreated clones. Frequencies of pGCcos3neo-mediated transfer of neo gene-encoded antibiotic resistance into B78H1 can reach 10(-2) in response to treatment with as little as 15 ng plasmid/ml coprecipitate/dish. B78H1 cells readily give rise to "secondary" transferents for the neo gene after treatment with DNA from a "primary" B78H1 neo transferent. This gene transfer system has potential applications for study of regulation of melanoma and neural crest differentiation and malignancy.

Animals↗
PubMed2007

Somatic sequence alterations in twenty-one genes selected by expression profile analysis of breast carcinomas.

INTRODUCTION: Genomic alterations have been observed in breast carcinomas that affect the capacity of cells to regulate proliferation, signaling, and metastasis. Re-sequence studies have investigated candidate genes based on prior genetic observations (changes in copy number or regions of genetic instability) or other laboratory observations and have defined critical somatic mutations in genes such as TP53 and PIK3CA. METHODS: We have extended the paradigm and analyzed 21 genes primarily identified by expression profiling studies, which are useful for breast cancer subtyping and prognosis. This study conducted a bidirectional re-sequence analysis of all exons and 5', 3', and evolutionarily conserved regions (spanning more than 16 megabases) in 91 breast tumor samples. RESULTS: Eighty-seven unique somatic alterations were identified in 16 genes. Seventy-eight were single base pair alterations, of which 23 were missense mutations; 55 were distributed across conserved intronic regions or the 5' and 3' regions. There were nine insertion/deletions. Because there is no a priori way to predict whether any one of the identified synonymous and noncoding somatic alterations disrupt function, analysis unique to each gene will be required to establish whether it is a tumor suppressor gene or whether there is no effect. In five genes, no somatic alterations were observed. CONCLUSION: The study confirms the value of re-sequence analysis in cancer gene discovery and underscores the importance of characterizing somatic alterations across genes that are related not only by function, or functional pathways, but also based upon expression patterns.

Breast Neoplasms↗
PubMed2005

The effect of benzo(a)pyrene on porcine urinary bladder epithelial cells analyzed for the expression of selected genes and cellular toxicological endpoints.

Consumption of tobacco products is the most relevant risk factor for the development of bladder cancer beside occupational contributions. In order to investigate mechanisms of tobacco smoke components in bladder carcinogenesis we have introduced a primary epithelial cell culture system derived from porcine urinary bladder as a suitable representative for the corresponding human tissue under physiological conditions. Two independent readouts were selected as markers for genotoxic events. Changes in the expression level of several toxicologically relevant genes should serve as indicators for early response, while classical genotoxic endpoints monitored manifested damages. Here, we present the first results of our study with benzo(a)pyrene (BaP) as a member of polycyclic aromatic hydrocarbons (PAHs) found in tobacco smoke. Cells treated with BaP show a dramatic increase in the expression of CYP1A1 that appears to be both indicator of and contributor for BaP toxicity. Genes coding for other proteins relevant in xenobiotic metabolism, signal transduction or tumor suppression show moderate effects or no enhancement of their expression levels. Comet assay and micronucleus test did show a significant, dose-dependent increase in DNA damages or aberrations after cell division. While these effects are conforming to the response at the mRNA expression level, they are less pronounced and require rather higher dosages of the chemical.

Air Pollutants↗
PubMed2002

Selective gene transfer into primary human gastric tumors using epithelial cell adhesion molecule-targeted adenoviral vectors with ablated native tropism.

Currently, application of adenoviral vectors (AdV) in gastric cancer gene therapy would be improved by increases in the specificity of transduction. Previously, we found that epithelial cell adhesion molecule (EpCAM) was expressed on gastric tumors but not on gastric epithelium. In this study, we evaluated doubly-ablated AdV lacking native binding ability together with bispecific single-chain antibodies targeted toward EpCAM for gene therapy of gastric cancer. Specific binding to EpCAM augmented the gene transfer efficiency of doubly-ablated AdV on gastric cancer cell lines up to 144-fold, reaching levels similar to or exceeding those achieved with native AdV. In contrast, EpCAM-targeted doubly-ablated AdV-mediated gene transfer into an EpCAM-negative cell line was reduced 38-fold compared with transduction by native AdV. Most importantly, EpCAM-targeted doubly-ablated AdV showed selectivity for primary human gastric tumors versus the surrounding nonneoplastic gastric mucosa of the same patients and normal liver tissue samples. Targeting these doubly-ablated AdV toward EpCAM resulted in similar transduction efficiency as obtained with native AdV for EpCAM-expressing primary human gastric tumors, whereas transduction of gastric epithelium and liver tissue was reduced at least 10-fold. This study thus indicates that application of EpCAM-targeted doubly-ablated AdV for gastric cancer gene therapy results in a favorable tumor-over-normal tissue transduction ratio.

Adenocarcinoma↗
PubMed1996

Regulation of T-cell death genes: selective inhibition of FasL- but not Fas-mediated function.

Activation-induced cell death (AICD) requires coexpression of Fas and FasL. Hybridoma T-cells express detectable FasL mRNA 3 to 5 hr after culture in anti-CD3-coated wells. High and steady expression of FasL mRNA was observed after 8-10 hr of activation. Expression of FasL cytotoxicity and AICD is consistent with the time-course of FasL mRNA induction. Fas-Ig was effective in inhibiting AICD when added no later than 5 hr after activation, but was ineffective when added after 8-10 hr of activation. These observations suggest that FasL gene activation is a critical step for AICD. By contrast, Fas was constitutively expressed and the time-course study did not support the idea that up-regulation of Fas is critical for AICD. The critical role of FasL gene activation for AICD was confirmed by studies using inhibitors of AICD. Dexamethasone (Dex) inhibited FasL induction and Fas up-regulation, but not basal Fas expression of hybridoma T-cells. All-trans retinoic acid (RA) inhibited FasL induction, but had little effect on Fas up-regulation. Both agents inhibited FasL cytotoxicity. The Fas-mediated death pathway distal to Fas/FasL interactions remained intact in the protected hybridoma T-cells. These results demonstrate that FasL gene activation, but not Fas up-regulation, is critical for AICD and that Dex and all-trans RA selectively inhibits FasL but not Fas function. The system may prove useful for the identification of critical factors regulating T-cell death genes. It may also serve as a useful system to study gene regulation in AICD-dependent phenomena.

Animals↗
PubMed2002

Identification of a new variant in the YMDD motif of the hepatitis B virus polymerase gene selected during lamivudine therapy.

A new hepatitis B virus variant selected during lamivudine treatment was detected, in which the methionine (rtM204) in the so-called YMDD motif in the C domain of the catalytic site of the polymerase gene was replaced by a serine (rtM204S). This change simultaneously resulted in a tyrosine-195 into valine variant (sY195V) in the surface protein HBsAg. The detection of this YSDD variant was initially observed, after an increase of HBV DNA levels, by sequencing of amplification products from day 586. A specific RFLP assay was developed that could identify 10% of YSDD-containing variants in the virus pool, which enabled detection of this new variant virus at day 506. However, by cloning several PCR products and sequencing individual recombinant clones, the mutation was first identified at day 477, before a significant increase of HBV DNA was observed in serum. The mutation was followed by a leucine to methionine change at position 180 (rtL180M). The consequences of this mutation for disease management and diagnostic strategies are discussed.

Alanine Transaminase↗
PubMed2006

Role of ETS transcription factors in the hypoxia-inducible factor-2 target gene selection.

Tumor hypoxia often directly correlates with aggressive phenotype, metastasis progression, and resistance to chemotherapy. Two transcription factors [hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha] are dramatically induced in hypoxic areas and regulate the expression of genes necessary for tumor adaptation to the conditions of low oxygen; however, the relative contribution of these factors is controversial. We used RNA interference-mediated inactivation of HIF-1alpha or HIF-2alpha followed by microarray analysis to identify genes specifically regulated by either HIF-1 or HIF-2 in hypoxia. We found that, in the MCF7 cell line, the vast majority of hypoxia-responsive genes (>80%) were dependent on the presence of HIF-1alpha. However, a small group of genes were preferentially regulated by HIF-2alpha. Promoter analysis for this group of genes revealed that all of them have putative binding sites for ETS family transcription factors, and 10 of 11 HIF-2alpha-dependent genes had at least one potential hypoxia-responsive element (HRE) in proximity to an ETS transcription factor binding site. Knockdown of ELK-1, the most often represented member of ETS family, significantly reduced hypoxic induction of the HIF-2alpha-dependent genes. Physical and functional interaction between ELK-1 and HIF-2alpha were supported by coimmunoprecipitation of these two proteins, luciferase reporter assay using CITED2 promoter, and binding of ELK-1 protein to the promoters of CITED2 and WISP2 genes in proximity to a HRE. These data suggest that the choice of the target genes by HIF-1 or HIF-2 depends on availability and cooperation of HIFs with other factors recognizing their cognate elements in the promoters.

Basic Helix-Loop-Helix Proteins↗
PubMed2002

Efficient and selective gene transfer into primary human brain tumors by using single-chain antibody-targeted adenoviral vectors with native tropism abolished.

The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeting adenoviral vectors toward tumor cells may improve cancer gene therapy procedures by providing augmented tumor transduction and decreased toxicity to normal tissues. Targeting requires both the complete abolition of native tropism and the addition of a new specific binding ligand onto the viral capsid. Here we accomplished this by using doubly ablated adenoviral vectors, lacking coxsackievirus-adenovirus receptor and alpha(v) integrin binding capacities, together with bispecific single-chain antibodies targeted toward human epidermal growth factor receptor (EGFR) or the epithelial cell adhesion molecule. These vectors efficiently and selectively targeted both alternative receptors on the surface of human cancer cells. Targeted doubly ablated adenoviral vectors were also very efficient and specific with primary human tumor specimens. With primary glioma cell cultures, EGFR targeting augmented the median gene transfer efficiency of doubly ablated adenoviral vectors 123-fold. Moreover, EGFR-targeted doubly ablated vectors were selective for human brain tumors versus the surrounding normal brain tissue. They transduced organotypic glioma and meningioma spheroids with efficiencies similar to those of native adenoviral vectors, while exhibiting greater-than-10-fold-reduced background levels on normal brain explants from the same patients. As a result, EGFR-targeted doubly ablated adenoviral vectors had a 5- to 38-fold-improved tumor-to-normal brain targeting index compared to native vectors. Hence, single-chain targeted doubly ablated adenoviral vectors are promising tools for cancer gene therapy. They should provide an improved therapeutic index with efficient tumor transduction and effective protection of normal tissue.

Adenoviridae↗
PubMed2006

Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula.

The identification of the molecular polymorphisms giving rise to phenotypic trait variability-both quantitative and qualitative-is a major goal of the present agronomic research. Various approaches such as positional cloning or transposon tagging, as well as the candidate gene strategy have been used to discover the genes underlying this variation in plants. The construction of functional maps, i.e. composed of genes of known function, is an important component of the candidate gene approach. In the present paper we report the development of 63 single nucleotide polymorphism markers and 15 single-stranded conformation polymorphism markers for genes encoding enzymes mainly involved in primary metabolism, and their genetic mapping on a composite map using two pea recombinant inbred line populations. The complete genetic map covers 1,458 cM and comprises 363 loci, including a total of 111 gene-anchored markers: 77 gene-anchored markers described in this study, 7 microsatellites located in gene sequences, 16 flowering time genes, the Tri gene, 5 morphological markers, and 5 other genes. The mean spacing between adjacent markers is 4 cM and 90% of the markers are closer than 10 cM to their neighbours. We also report the genetic mapping of 21 of these genes in Medicago truncatula and add 41 new links between the pea and M. truncatula maps. We discuss the use of this new composite functional map for future candidate gene approaches in pea.

Chromosome Mapping↗
PubMed1998

Identification of a gene selectively expressed in the brain, which encodes a putative transmembrane protein and a soluble cytoplasmic isoform.

Subtractive cloning procedures led to the identification of a variety of transcripts expressed in mammalian brain. However, little is known about the encoded proteins and the regulation of gene expression. Here, we describe the isolation and characterisation of a single-copy gene (83.5) of 21.7 kb which is specifically expressed in porcine brain. In situ hybridisation and immunohistochemistry experiments showed a distinct pattern of gene expression in neuronal cell types in different parts of the brain. The gene contains two mini exons, confirming neural-specific expression. cDNA cloning experiments revealed two species of mRNA differing in their 5'-regions. These transcripts are generated by two distinct transcription start sites that are under the control of different potential promoter regions as shown by primer-extension experiments. The amino acid sequences of the deduced proteins predict that one mRNA species encodes a novel type-I transmembrane protein, whereas the other transcript encodes only a part of its cytoplasmic domain. In Western-blot experiments, we detected two proteins of the predicted size and cellular localisation in porcine brain. The precise function of these proteins remains to be determined. However, our findings suggest that they may be generated by alternative promoter usage, leading to the expression of a membrane protein and its truncated cytoplasmic isoform.

Amino Acid Sequence↗
PubMed2003

Susceptible MHC alleles, not background genes, select an autoimmune T cell reactivity.

To detect and characterize autoreactive T cells in diabetes-prone NOD mice, we have developed a multimeric MHC reagent with high affinity for the BDC-2.5 T cell receptor, which is reactive against a pancreatic autoantigen. A distinct population of T cells is detected in NOD mice that recognizes the same MHC/peptide target. These T cells are positively selected in the thymus at a surprisingly high frequency and exported to the periphery. They are activated specifically in the pancreatic LNs, demonstrating an autoimmune specificity that recapitulates that of the BDC-2.5 cell. These phenomena are also observed in mouse lines that share with NOD the H-2g7 MHC haplotype but carry diabetes-resistance background genes. Thus, a susceptible haplotype at the MHC seems to be the only element required for the selection and emergence of autoreactive T cells, without requiring other diabetogenic loci from the NOD genome.

Alleles↗
PubMed1988

Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.

Gene targeting--homologous recombination of DNA sequences residing in the chromosome with newly introduced DNA sequences--in mouse embryo-derived stem cells promises to provide a means to generate mice of any desired genotype. We describe a positive nd negative selection procedure that enriches 2,000-fold for those cells that contain a targeted mutation. The procedure was applied to the isolation of hprt- and int-2- mutants, but it should be applicable to any gene.

Animals↗
PubMed1993

Variable region gene selection of immunoglobulin G-expressing B cells with specificity for a defined epitope on type II collagen.

Immunization with type II collagen (CII) induces collagen-induced arthritis (CIA) in animals, and B cells reactive with CII are involved in the induction and manifestation of the disease. In this study, B cell hybridomas producing IgG antibodies specific for a major epitope on mouse CII (the "C1" epitope, amino acid 316-333), were isolated 11 days after immunization from draining lymph nodes in DBA/1 mice. Injection into neonatal mice of purified and biotinylated monoclonal antibodies binding the C1 epitope led to a specific binding to joint cartilage, demonstrating that the antibodies interact with native antigen in vivo. cDNA sequencing of the B cell clones revealed that they all expressed the same combination of a variable heavy chain (VH J558 family) and light chain (V kappa 21 family) germ-line gene, apparently lacking somatic mutations. The presence of isotype-switched B cells expressing a certain combination of V genes encoding antibodies that bind epitopes in vivo, indicates that this B cell population has been peripherally selected.

Amino Acid Sequence↗
PubMed2004

Guideline to reference gene selection for quantitative real-time PCR.

Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and beta-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that "Classical" reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells.

Base Sequence↗
PubMed1996

DNA repair domains within a human gene: selective repair of sequences near the transcription initiation site.

We describe a new form of DNA repair heterogeneity along the genome. The repair rate of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured at single nucleotide resolution along the promoter and transcribed sequences of the human JUN gene in UV-irradiated diploid fibroblasts. The promoter of this gene contains an array of sequence-specific transcription factors located between nucleotides -200 and -50 relative to the major transcription start site. These sequences are repaired slowly; at many sites >50% of the CPDs are left unrepaired after 24 h. However, repair rates are 10-fold faster near the transcription initiation site. This very fast repair is seen on both DNA strands between nucleotides -40 and +100 where at most positions >90% of the dimers are repaired within 4 h. There is a general gradient of repair efficiency of the transcribed DNA strand with faster repair within the 5'-end and diminished repair towards the 3'-end of the gene. The fast repair rates seen near the transcription initiation site may be explained by increased local concentrations of DNA repair factors that are associated with general transcription factors (e.g. TFIIH) functioning in transcription initiation. This domain-specific DNA repair may aid in maintaining transcription initiation of essential genes after DNA damage.

Base Sequence↗
PubMed2005

Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression.

Stabilized plasmid lipid particles (SPLP) consist of a single copy of DNA surrounded by a lipid bilayer. The particles are small ( approximately 100 nm), stable, monodisperse and have a low surface charge. A diffusible polyethylene glycol (PEG) coating attached to a lipid anchor is critical to the SPLP's functionality. The PEG-lipid exchanges out of the bilayer at a rate determined by the size of the lipid anchor. Here we show that SPLP can be prepared using a series of PEG-diacylglycerol lipids (PEG-S-DAGs). SPLP were prepared incorporating PEG-dimyristoylglycerol (C14), PEG-dipalmitoylglycerol (C16) or PEG-distearoylglycerol (C18) and the rate of PEG-lipid diffusion from the bi-layer determined using a FRET assay. SPLP pharmacokinetics confirm a correlation between the stability of the PEG-lipid component and circulation lifetime. PEG-S-DAGs with longer lipid anchors yield more stable SPLP particles with longer circulation half-lives yielding an increase in tumor delivery and gene expression. PEG-distearoylglycerol (C18) containing SPLP bypass so-called 'first pass' organs, including the lung, and elicit levels of gene expression in distal tumor tissue 100- to 1000-fold greater than that observed in any other tissue. The incorporation of PEG-S-DAG in SPLP confirms that small size, low surface charge and extended circulation lifetimes are prerequisite to the accumulation and tumor selective expression of plasmid DNA following systemic administration.

Diglycerides↗