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At least 649 records · Page 36Linked to original sources
PubMed1995

In vivo regulation of interleukin-2 receptor alpha gene transcription by the coordinated binding of constitutive and inducible factors in human primary T cells.

IL-2R alpha transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. To analyse the mechanisms used to activate IL-2R alpha transcription as well as those used to block it in non-expressing cells, we determined the protein-DNA interactions at the IL-2R alpha locus in three different cell types using the DMS/LMPCR genomic footprinting method. CD25/IL-2R alpha can be efficiently induced in primary human T cells since approximately 100% express this gene when receiving an appropriate combination of mitogenic stimuli. To understand why IL-2R alpha is not expressed in other haematopoietic cell types, we analysed BJAB B lymphoma cells which do not express the IL-2R alpha gene and contain constitutively active nuclear NF-kappa B. Primary fibroblasts from embryo and adult skin were selected to examine the mechanisms that may be used to keep the IL-2R alpha gene inactive in non-haematopoietic cells. The three main results are: (i) the stable in vivo occupancy of IL-2R alpha kappa B element in resting T cells, most probably by constitutive NF-kappa B p50 homodimer that could impair SRF binding to the flanking SRE/CArG box; (ii) its inducible occupancy by NF-kappa B p50-p65 associated with the binding of an SRE/CArG box DNA-binding factor upon mitogenic stimulation; and (iii) a correlation between the precommitment of T cells to activation and the presence of stable preassembled protein-DNA complexes in contrast with the bare IL-2R alpha locus in non-T cells.

Antineoplastic Agents↗
PubMed2003

Inhibition of type I procollagen production by tRNAVal CTE-HSP47 ribozyme.

BACKGROUND: Fibrosis characteristically occurs in the advanced stages of chronic inflammatory diseases, occasionally as the primary lesion, and frequently determines the disease prognosis. Fibrotic lesions consist mostly of collagen, and therefore it may be possible to prevent or treat fibrosis by inhibiting collagen production. Of the currently available therapeutic approaches, however, none is sufficiently effective and specific for inhibition of collagen. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been reported to play a pivotal role in secretion of procollagen molecules. Therefore, we have tried to suppress its function to inhibit these various types of collagen. METHODS: We have developed a novel type of ribozyme by ligating a hammerhead sequence to a tRNA(Val) promoter to facilitate displacing the ribozyme from nucleus to cytoplasm and to constitutive transport element, a binding motif of helicase which unwinds mRNA to render the target sequence on the mRNA accessible to the ribozyme. RESULTS: The ribozyme thus constructed showed strong activity to cleave HSP47 mRNA and suppress the secretion of type I procollagen in the human primary fibroblast. CONCLUSION: We suggest applicability of this ribozyme as a new modality for antifibrosis therapy.

Antisense Elements (Genetics)↗
PubMed2005

Type XIII collagen expression is induced during malignant transformation in various epithelial and mesenchymal tumours.

Little information is available on the expression of transmembrane type XIII collagen in human diseases. The present study has investigated the expression of this collagen in cancer, in particular during malignant transformation. By combining the tissue microarray technique with in situ hybridization, a consistent pattern of clearly increased type XIII collagen mRNA expression was found in the stromal compartment of epithelial tumours and throughout mesenchymal tumours. Slightly elevated mRNA expression was observed in dysplastic samples and in malignant epithelial cells. It is also demonstrated that factors secreted into the culture medium by tumour cells, in particular the growth factor TGF-beta, contribute to the induction of type XIII collagen expression, and trigger concomitantly a profound phenotypic and morphological transition of cultured primary fibroblasts. Reciprocally, type XIII collagen may alter the growth milieu of malignant cells as the soluble type XIII collagen ectodomain influenced the adherence and spreading of cells cultured on vitronectin-rich matrix. It is proposed that malignant transformation stimulates the expression of type XIII collagen, particularly in the tumour stroma and to a lesser extent in the epithelium, and that this high type XIII collagen expression may contribute to tumour progression and behaviour by modulating cell-matrix interactions.

Cell Adhesion↗
PubMed1995

3-Aminobenzamide protects cells from UV-B-induced apoptosis by acting on cytoskeleton and substrate adhesion.

3-aminobenzamide (3-ABA) is an inhibitor of poly-(ADP-ribose)-polymerase, an enzyme involved in numerous subcellular processes, including cell death. Recently, a target effect of the drug on some cytoskeletal elements has also been described (Malorni et al., Biochem. Biophys. Res. Commun. 202: 915-922, 1994). In this study we evaluated the ability of 3-ABA to interfere with UV-B ray-induced apoptosis in cells selected for their cytoskeletal features and their different capability to adhere to the substrate. Human melanoma (M14) and epithelial (A431) cell lines and murine primary fibroblastic cultures (MFC) were studied. Our results indicate that cytoskeleton is indeed an important cellular target of 3-ABA, which can prevent apoptotic cell death by UV-B through a specific effect on the adhesion properties of the cells. Indeed, an inverse correlation was observed between sensitivity to UV-B-induced apoptosis (M14 > A431 > MFC) and substrate adhesion (MFC > A431 > M14). The potential relevance of these observations to understand the possible relationships among apoptosis, cytoskeletal functions and substrate adhesion is discussed.

Animals↗
PubMed1997

Efficient gene transduction by Epstein-Barr-virus-based vectors coupled with cationic liposome and HVJ-liposome.

We show here a novel non-viral strategy to transduce human cells by using an EBV-based vector system. The EBV-based vectors, the plasmid vectors carrying EBV oriP (origin for plasmid replication) and EBNA (EBV nuclear antigen) 1 gene from EBV genome, were combined with 2 gene delivery systems, i.e., cationic liposome and HVJ-liposome. By both methods, EBV-based vectors could be more efficiently transfected into HeLa cells than non-EBV, conventional plasmid vectors. When human primary fibroblasts were transfected, EBV-based vectors coupled with cationic liposome but HVJ-liposome resulted in successful gene transduction, while human bone marrow cells were transduced with both HVJ-liposome- and cationic liposome-EBV vectors. These results suggest the potential applications of the EBV-based vector system for gene therapy.

Bone Marrow Cells↗
PubMed1993

DNA methylation is not involved in growth regulation of gene expression of proliferating cell nuclear antigen.

Expression of proliferating cell nuclear antigen (PCNA) gene is growth regulated. By Southern blot analysis with restriction enzymes such as HpaII and MspI, no change of DNA methylation state in this gene was detected during rat liver regeneration. There is only one HpaII site located in the promoter region of rat PCNA gene and this HpaII site was found to be demethylated in both normal and regenerating livers. Human hepatoma cell lines Hep G2 and Hep 3B were used to study the relation of PCNA gene expression and the DNA methylation. There are 16 HpaII sites in human PCNA gene, and according to the HpaII and MspI restriction patterns, many of the HpaII sites were methylated in vivo. Upon serum stimulation of quiescent cells, no change of DNA methylation state in the HpaII and HhaI sites was found. Demethylation by the methylation inhibitor, 5-azacytidine, did not affect the expression of PCNA gene in the hepatoma cells. With the human primary fibroblasts, Y2, the demethylation by 5-azacytidine did not seem to change the growth dependence of PCNA gene expression. This is consistent with the observation that the expression of PCNA gene of some cultured cell lines such as CHO.K1, in which the PCNA gene was unmethylated, showed growth dependence. Also, no variation in methylation pattern of PCNA gene was found among the different rat tissues in which the expression of PCNA varies. Therefore, we conclude that DNA methylation is not involved in growth regulation of the PCNA gene expression.

Animals↗
PubMed1999

An inhibitor of nuclear export activates the p53 response and induces the localization of HDM2 and p53 to U1A-positive nuclear bodies associated with the PODs.

Leptomycin B is a cytotoxin which directly interacts with and inhibits the action of CRM1, an essential mediator of the nuclear exit of proteins containing nuclear export signals (NES) of the HIV1 REV type. We show that addition of leptomycin B to human primary fibroblasts increased the levels of the p53 tumor suppressor protein. This was accompanied by the induction of p53-dependent transcriptional activity in cultured cells and an increase in the levels of the products of two p53-responsive genes, the p21(CIP1/WAF1) and HDM2 proteins. Leptomycin B induced the accumulation of p53 and HDM2 in the nucleus and the appearance of discrete nuclear aggregates containing both proteins. It has been reported that the transcriptional activity of p53 is modulated by its interaction with the HDM2 protein which also targets p53 for rapid degradation. Using a model cell line conditionally expressing MDM2, the murine analogue of HDM2, we present evidence indicating that leptomycin B abrogates MDM2's role in p53 degradation and that the accumulation of p53 in distinct nuclear bodies is mediated by MDM2. Since HDM2 has recently been shown to contain a functional NES of the REV type, the most likely explanation for our results is that the effect of leptomycin B on HDM2 and p53 is due to the inhibition of nuclear export. The ability to visualize sites where p53 and HDM2 colocalize provides a new approach to study the association between the two proteins in vivo. These p53/HDM2-positive nuclear foci were found to also contain the U1A snRNP A and to be juxtaposed to the PML oncogenic domains.

Biological Transport↗
PubMed2000

Independent role of p38 and ERK1/2 mitogen-activated kinases in the upregulation of matrix metalloproteinase-1.

Matrix metalloproteinase-1 (MMP-1) plays an important role in the degradation of extracellular matrix components under several physiological and pathological conditions. The expression of this protease is upregulated by mitogenic growth factors and proinflammatory cytokines, which have been shown to activate different sets of mitogen-activated protein (MAP) kinase pathways. Here we provide evidence that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) or the p38 MAP kinase pathway is sufficient to induce transcription from the MMP-1 promoter in human primary fibroblasts, whereas modulation of mRNA stability seems to be of minor importance. Upregulation of MMP-1 expression by mitogenic or inflammatory stimuli is blocked by specific small molecular weight inhibitors of the ERK pathway or the p38 pathway, respectively, and constitutively active kinases within the ERK1/2 pathway (MEKK1, MEK1) or the p38 pathway (ASK1, MEKK1, MKK3) are potent activators of the MMP-1 promoter. The current study provides evidence that distinct extracellular signals leading to upregulation of MMP-1 expression in fibroblasts are relayed independently through different MAP kinase pathways and are integrated at the level of the promoter.

Cyclooxygenase 2↗
PubMed1993

Vertical integration of cosmid and YAC resources for interval mapping on the X-chromosome.

The vertical integration of cosmid and yeast artificial chromosome (YAC) resources is of particular importance in the development of high-resolution maps of selected regions of the human genome. A resource of approximately 95,000 cosmids constructed using DNA from primary fibroblasts of karyotype 49,XXXXX was validated by detailed characterization of a 200-kb cosmid contig spanning exons 8-20 of the dystrophin gene. This resource was used to construct contigs in 0.65 Mb of Xq26 by hybridization of gel-purified YAC DNA to high-density gridded arrays of the cosmid library; positive cosmids were overlapped by finger-printing. Contigs were oriented and ordered relative to existing YACs in the region using cross-hybridization. The overlaps between a representative set of cosmids define 54 intervals of 5-20 kb and were used to construct a high-resolution cosmid interval map of the region, locating markers, dinucleotide repeats, and candidate CpG islands. This approach can be applied rapidly to large regions of the genome and without recourse to subcloning of individual YACs.

Aneuploidy↗
PubMed2000

Protein binding by the 3' untranslated region of alpha-striated tropomyosin.

Tropomyosin is a component protein of the thin filament system in striated muscle, regulating the interaction between actin and myosin. The 3' untranslated region of the alpha-striated tropomyosin gene (TM UTR) induces muscle differentiation when expressed in primary fibroblasts, but the mechanism has not been defined. We hypothesize that fibroblasts utilize resident proteins to effect this response, perhaps by TM UTR binding to protein(s). In order to facilitate identification of protein(s) involved in mediating this differentiation response, we investigated the potential for this sequence to bind to cellular protein utilizing electrophoretic mobility gel shifting analysis (EMSA) with and without UV cross-linking. Under very specific conditions (including pH, KCl, and Mg concentration and extent of phosphorylation of protein), the TM UTR is able to bind protein in cells that differentiate upon TM UTR expression. Protein binding is significantly more extensive in cytoplasmic than nuclear protein preparations. Secondary structure of the RNA probe facilitates protein binding. The molecular masses of bound proteins are approximately 42 and 115 kDa under basal conditions. EMSA analysis of extract from cultured skeletal muscle confirms that protein binding by the TM UTR occurs in this cell type, and is more extensive in less differentiated cells. The demonstration of highly regulated protein binding by the TM UTR raises the possibility that this sequence may cause differentiation by binding to endogenous proteins, and further that this sequence may play a role in normal differentiation. Identification of proteins bound by the TM UTR will be necessary to completely define the mechanism by which it causes differentiation.

3' Untranslated Regions↗
PubMed2002

Transfection screening for defects in the PCCA and PCCB genes encoding propionyl-CoA carboxylase subunits.

Propionic acidemia can result from mutations in the PCCA or PCCB genes encoding the alpha and beta subunits, respectively, of propionyl-CoA carboxylase. We have developed a method based on complementation of the enzyme defect using a lipid-mediated transient transfection of the normal human PCCA or PCCB cDNA into primary fibroblasts. We demonstrate the reliability of this method for identification of the defective PCC gene in order to unequivocally approach the mutational analysis in the corresponding PCCA and PCCB genes.

Carboxy-Lyases↗
PubMed2001

Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison.

Recombinant adeno-associated (rAAV) viral vectors hold great therapeutic potential for human diseases. However, a relatively small packaging capacity (less than 5 kb) has limited the application of rAAV for certain diseases such as cystic fibrosis and Duchenne muscular dystrophy. Here we compared two mechanistically distinct approaches to overcome packaging restraints with rAAV vectors. The trans-splicing approach reconstitutes gene expression from two independent rAAV vectors, each encoding unique, nonoverlapping halves of a transgene. This process requires intermolecular concatamerization and subsequent splicing between independent vectors. A distinct overlapping vector approach uses homologous recombination between overlapping regions in two independent vectors. Using the beta-galactosidase gene as template, trans-splicing approaches were threefold (in primary fibroblasts) and 12-fold (in muscle tissue) more effective in generating full-length transgene products than the overlapping vector approach. Nevertheless, the efficiency of trans-splicing remained moderate at approximately 4.3% (for muscle) and 7% (for fibroblasts) of that seen with a single vector encoding the full-length transgene. The efficiency of trans-splicing was augmented 1185-fold by adenoviral E4, but not E2a, gene products. This augmentation was much less pronounced with the overlapping vectoring approach (12-fold). Trans-splicing and overlapping vector approaches are two viable alternatives to expand rAAV packaging capacity.

Adenovirus E2 Proteins↗
PubMed2002

In vivo hepatic adenoviral gene delivery occurs independently of the coxsackievirus-adenovirus receptor.

Systemic administration of adenoviral vectors leads to a widespread distribution of vector. Therefore, targeting of adenoviral vectors to specific tissues or cell types will require methods to ablate the normal tropism of the vector simultaneously with the introduction of new receptor specificities. To inhibit native receptor binding, we mutated residues in the AB loop of the adenovirus type 5 (Ad5) fiber. We genetically incorporated the S408E-P409A mutation, referred to as KO1, into the adenoviral genome alone or in combination with an RGD-targeting ligand in the HI loop of fiber. Transduction experiments confirmed that the KO1 mutation results in a significant reduction in fiber-dependent gene transfer on A549 and primary fibroblast cells that could be restored via the RGD-targeting ligand. Competition transduction experiments verified the receptor-binding properties of each vector on A549 and hepatocytes in vitro. Unexpectedly, in mice systemic delivery of the vector containing the KO1 mutation resulted in efficient liver transduction that was localized specifically to hepatocytes. We confirmed these results in three different mouse strains, indicating that hepatic adenoviral gene transfer may be independent of the coxsackievirus-adenovirus receptor and that in vivo retargeting will require further viral capsid modifications to generate a fully detargeted adenoviral vector upon which to introduce new tropisms.

Adenoviruses, Human↗
PubMed1999

Chromosome missegregation and trisomy 21 mosaicism in Alzheimer's disease.

A connection between Alzheimer's disease (AD) and Down syndrome (trisomy 21) is indicated by the fact that all Down syndrome individuals develop Alzheimer's disease neuropathology by the 4th decade of life. Previous studies have examined the frequency of aneuploidy and other chromosomal defects in cells from familial Alzheimer's disease (FAD) patients, with varying results. To investigate the possibility that a specific type of aneuploidy--trisomy 21 mosaicism--may contribute to Alzheimer's disease, we used quantitative fluorescence in situ hybridization to measure the number of trisomy 21 cells in primary fibroblast cultures from AD and unaffected subjects. The 27 AD cultures, including 15 that were derived from individuals carrying FAD mutations in presenilin 1 or 2, exhibited a significant approximately twofold increase in the number of trisomy 21 cells compared to 13 control cultures. A small double-hybridization experiment suggested that the aneuploidy in AD cells was not limited to chromosome 21 but extended at least to chromosome 18 as well. In a parallel study, the endogenous presenilin proteins in fibroblasts were localized to the centrosomes, the nuclear envelope, and its associated interphase kinetochores. Together these results indicate that the presenilin proteins may be involved in mitosis and that FAD mutations in the presenilin genes may predispose to chromosome missegregation (nondisjunction). The data reported here also suggest that trisomy 21 mosaicism may contribute to other forms of AD that are not caused by a presenilin mutation.

Alleles↗
PubMed1999

Structural analysis of adeno-associated virus transduction circular intermediates.

Recombinant adeno-associated virus (rAAV) has recently been demonstrated to form circular intermediates following transduction in muscle tissue and cell lines. Although restriction enzyme and Southern blot analysis has revealed a consistent monomer and multimer head-to-tail conformation, detailed structural sequence analysis has been lacking due to the high secondary structure of the ITR arrays. To gain further insight into potential mechanisms by which AAV circular genomes are formed from linear single-stranded viral DNA, we have performed chemical sequencing of ITR arrays within seven circular intermediates independently isolated from primary fibroblasts and Hela cells. Results from these studies demonstrated several types of circular intermediates with mosaic ITR elements flanked by two D sequences. The most predominant form consisted of a structure similar to that of previously generated AAV double-D plasmids, with one complete ITR flanked by two D-region elements. However, intermediately deleted ITR arrays with more than one complete ITR were also seen. Based on this structural information, we have proposed a model for formation of AAV circular intermediates by recombination/ligation between ITR ends of panhandle single-stranded AAV genomes.

Base Sequence↗
PubMed2001

Critical role for SV40 small-t antigen in human cell transformation.

Defining the ability of simian virus 40 (SV40) to transform human cells has become of even greater importance with the increased understanding that this virus may play a role in some human malignancies. This report documents the requirement for viral small-t (ST) antigen in large-T (LT)-driven transformation of primary fibroblasts, a requirement that cannot be met by a well-known oncogene, c-Ha-ras (EJ-ras), which can cooperate with LT in rodent systems. The cellular gene telomerase is not essential for transformation, although transformed clones are not immortal without it. Similarly, an immortal mesothelial cell line has been developed using LT and telomerase. Immortalized mesothelial cells are morphologically normal, but can be transformed by introduction of ST, or ST + ras, but not by ras alone. It is likely that ST will be required along with LT for transformation of most human cell types.

Antigens, Polyomavirus Transforming↗
PubMed1990

Partial complementation of the Fanconi anemia defect upon transfection by heterologous DNA. Phenotypic dissociation of chromosomal and cellular hypersensitivity to DNA cross-linking agents.

Transfectants obtained by mouse DNA-mediated gene transfer in Fanconi anemia (FA) primary fibroblasts from the genetic complementation groups A and B were examined for the frequencies of chromosomal aberrations and cytotoxicity following treatments by cross-linking agents. Cells from group A (FA 150), which is the most sensitive to such agents, are partially corrected for both the chromosomal and cellular hypersensitivity to 8-methoxypsoralen photoaddition. In contrast, after treatment with mitomycin C (MMC), only the chromosomal sensitivity is re-established to a near normal level. The opposite is true for FA group B cells (FA 145), i.e. cell survival to MMC is partially corrected, whereas the frequency of MMC-induced chromosomal aberration remains close to that of the untransfected cells. The partial phenotypic correction of the two end points examined is interpreted as indicating either a gene dosage effect or the necessity of introducing more than one gene type in order to achieve complete recovery of a normal phenotype. The phenotypic dissociation between the clastogenic and cellular hypersensitivity to cross-linking agents may offer the opportunity of isolating separately the responsible gene(s) by conventional rescue techniques.

Animals↗
PubMed1995

The bovine bivariate flow karyotype and peak identification by chromosome painting with PCR-generated probes.

A bovine bivariate flow karyotype has been established from a primary fibroblast cell culture carrying a 4;10 Robertsonian translocation. From 27 to 36 populations could be resolved by flow cytometry although the anticipated number was 31. Separation of chromosomal pairs into two populations explains this high resolution and confirms the high level of heteromorphism previously observed. We used a PARM-PCR (Priming Authorizing Random Mismatches) procedure for the production of paint probes from flow-sorted chromosome fractions. These probes were used for chromosome identification by fluorescence in situ hybridization (FISH) on R-banded metaphase spreads. We present the localization of all the bovine chromosome types on the flow karyotype. Twenty-two chromosome types including the translocated chromosome were sorted as pure fractions.

Animals↗