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At least 1,063 records · Page 59Linked to original sources
PubMed2003

Retinoid target gene activation during induced tumor cell differentiation: human embryonal carcinoma as a model.

Many agents that exhibit chemopreventive activity are able to mediate a differentiation response in premalignant and malignant tissues. One of the most widely studied classes of tumor differentiation agents is the retinoids. There is rapidly evolving evidence for beneficial retinoid actions in the prevention or treatment of clinical tumors. However, the use of retinoids in the clinic is limited by acquired resistance and toxicity, especially when administered chronically in preventive strategies. Although retinoids are known to regulate gene transcription by activating retinoid receptors, the identity of the target genes that mediate the beneficial effects of retinoids are largely unknown. Here we review a useful model of retinoid-induced tumor cell differentiation: human embryonal carcinoma. The pluripotent nature and ease of use make human embryonal carcinoma cells a valuable and practical complement to human embryonic stem cells as an in vitro model of early human development. In addition, retinoid treatment of human embryonal carcinoma is an important model of induced tumor cell differentiation because retinoids cause the reversal of the malignant phenotype coincident with terminal neuronal differentiation. We have used both de novo and candidate approaches with this system in an effort to uncover critical downstream targets of retinoid receptors during differentiation induction.

Carcinoma, Embryonal↗
PubMedDate not supplied

High-resolution genomic and expression profiling reveals 105 putative amplification target genes in pancreatic cancer.

Comparative genomic hybridization (CGH) studies have provided a wealth of information on common copy number aberrations in pancreatic cancer, but the genes affected by these aberrations are largely unknown. To identify putative amplification target genes in pancreatic cancer, we performed a parallel copy number and expression survey in 13 pancreatic cancer cell lines using a 12,232-clone cDNA microarray, providing an average resolution of 300 kb throughout the human genome. CGH on cDNA microarray allowed highly accurate mapping of copy number increases and resulted in identification of 24 independent amplicons, ranging in size from 130 kb to 11 Mb. Statistical evaluation of gene copy number and expression data across all 13 cell lines revealed a set of 105 genes whose elevated expression levels were directly attributable to increased copy number. These included genes previously reported to be amplified in cancer as well as several novel targets for copy number alterations, such as p21-activated kinase 4 (PAK4), which was previously shown to be involved in cell migration, cell adhesion, and anchorage-independent growth. In conclusion, our results implicate a set of 105 genes that is likely to be actively involved in the development and progression of pancreatic cancer.

Cell Line, Tumor↗
PubMed2002

Tumor regression by targeted gene delivery to the neovasculature.

Efforts to influence the biology of blood vessels by gene delivery have been hampered by a lack of targeting vectors specific for endothelial cells in diseased tissues. Here we show that a cationic nanoparticle (NP) coupled to an integrin alphavbeta3-targeting ligand can deliver genes selectively to angiogenic blood vessels in tumor-bearing mice. The therapeutic efficacy of this approach was tested by generating NPs conjugated to a mutant Raf gene, ATPmu-Raf, which blocks endothelial signaling and angiogenesis in response to multiple growth factors. Systemic injection of the NP into mice resulted in apoptosis of the tumor-associated endothelium, ultimately leading to tumor cell apoptosis and sustained regression of established primary and metastatic tumors.

Adenosine Triphosphate↗
PubMed2006

E2F3 is the main target gene of the 6p22 amplicon with high specificity for human bladder cancer.

Amplification of 6p22 occurs in about 10-20% of bladder cancers and is associated with enhanced tumour cell proliferation. Candidate target genes for the 6p22 amplicon include E2F3 and the adjacent gene NM_017774. To clarify which gene is representing the main target, we compared the prevalence of the amplification and the functional role of both genes. Amplification of E2F3 and NM_017774 was analysed by fluorescence in situ hybridization on a bladder cancer tissue microarray composed of 2317 cancer samples. Both genes showed amplification in 104 of 893 (11.6%) interpretable tumours and were exclusively found co-amplified. Additional gene expression analysis by real-time polymerase chain reaction in 12 tumour-derived cell lines revealed that amplification of 6p22 was always associated with co-overexpression of E2F3 and NM_017774. Furthermore, RNA interference was used to study the influence of reduced gene expression on cell growth. In tumour cells with and without the 6p22 amplicon, knockdown of E2F3 always lead to unequivocal reduction of proliferation, whereas knockdown of NM_017774 was only capable to slow down cell proliferation in non-amplified cells. Our findings point out that E2F3 but not NM_017774 is driving enhanced proliferation of 6p22 amplified tumour cells. We conclude that E2F3 must be responsible for the growth advantage of 6p22 amplified bladder cancer cells.

Cell Proliferation↗
PubMed2002

Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination.

The genome sequence of Plasmodium falciparum, the causative agent of the most severe form of malaria in humans, rapidly approaches completion, but our ability to genetically manipulate this organism remains limited. Chromosomal integration has only been achieved following the prolonged maintenance of circularised episomal plasmids which selects for single crossover recombinants. It has not been possible to construct genetic deletions via double crossover recombination, presumably due to the low frequency of this event. We have used the Herpes simplex virus thymidine kinase gene and the Escherichia coli cytosine deaminase gene for negative selection of P. falciparum. Parasites were transformed with plasmids expressing the thymidine kinase and cytosine deaminase genes by positive selection for the human dihydrofolate reductase gene. Parasites expressing thymidine kinase are susceptible to the pro-drug ganciclovir while those expressing cytosine deaminase are sensitive to 5-fluorocytosine. Parental parasites were inherently resistant to these drugs. A significant 'bystander effect' was evident in cultures with either ganciclovir or 5-fluorocytosine. Positive and negative selection of the thymidine kinase transformants with both ganciclovir and WR99210 resulted in the selection of parasites containing a genetic deletion of the Pfrh3 gene, the first targeted double crossover deletions in P. falciparum. The use of negative selection for gene disruptions via double crossover recombination will dramatically improve our ability to analyse protein function and opens the possibility of using this strategy for a variety of gene deletion and modification experiments in the analysis of this important infectious agent.

Animals↗
PubMed1997

Gene targeting and instability of Agrobacterium T-DNA loci in the plant genome.

To develop a model system for studies of homologous recombination in plants, transgenic Nicotiana tabacum and Nicotiana plumbaginifolia lines were generated harbouring a single target T-DNA containing the negative selective codA gene encoding cytosine deaminase (CD) and the beta-glucuronidase (GUS) gene. Subsequently, the target lines were transformed with a replacement-type T-DNA vector in which the CD gene and the GUS promoter had been replaced with a kanamycin-resistance gene. For both Nicotiana species kanamycin-resistant lines were selected which had lost the CD gene and the GUS activity. One tobacco line was the result of a precise gene targeting event. However, most other lines were selected due to a chromosomal deletion of the target locus. The deletion frequency of the target locus varied between target lines, and could be present in up to 20% of the calli which were grown from leaf protoplasts. T-DNA transfer was not required for induction of the deletions, indicating that the target loci were unstable. A few lines were obtained in which the target locus had been deleted partially. Sequence analysis of the junctions revealed deletion of DNA sequences between microhomologies. We conclude that T-DNAs, which are stable during plant development as well as in transmission to the offspring, may become unstable during propagation in callus tissue. The relationships between callus culture, genetic instability and the process of T-DNA integration and deletion in the plant genome are discussed.

Base Sequence↗
PubMed1996

The involvement of brain-derived neurotrophic factor in hippocampal long-term potentiation revealed by gene targeting experiments.

Brain-derived neurotrophic factor (BDNF) is a member of the NGF gene family, which has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. The possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. To determine whether BDNF has a function in processes like long-term potentiation (LTP), we produced a strain of mice with a deletion in the coding sequence of the BDNF-gene. We then used hippocampal slices from these mice to investigate whether LTP is affected by this mutation. Mutant mice showed significantly weaker LTP in the CA1 region. The magnitude of the potentiation as well as the percentage of cases in which LTP could be induced successfully was clearly reduced whereas important pharmacological and morphological control parameters in the hippocampus of these animals were unaffected. Adenoviral vectors were used to re-express BDNF in acute slices of BDNF-knock-out mice. In most cases LTP could be rescued with this approach. These results suggest that BDNF has an important functional role in the expression of LTP in the hippocampus.

Animals↗
PubMed2003

Antiviral immune responses in gene-targeted mice expressing the immunoglobulin heavy chain of virus-neutralizing antibodies.

Two gene-targeted immunoglobulin heavy chain transgenic mouse strains, TgH(KL25) and TgH(VI10), expressing neutralizing specificities for lymphocytic choriomeningitis virus and vesicular stomatitis virus, respectively, have been generated. Three days after lymphocytic choriomeningitis virus infection, TgH(KL25) mice showed a thymus-independent neutralizing IgM response followed by thymus-dependent (TD) IgG. In contrast, WT mice mounted only a TD IgG response around day 80. These observations indicated that not only structural properties of the virus but also immunological parameters such as the frequency of B cells were indicative for the induction of thymus-independent versus TD Ig responses. Naïve vesicular stomatitis virusspecific Ig heavy chain transgenic mice displayed greatly elevated natural antibody titers. However, despite these high naïve titers, de novo activation of naïve CD4+ T and B cells was not blocked. Therefore, B cells giving rise to natural antibodies do not participate in virus-induced antibody responses.

Animals↗
PubMed2004

Macroarray analysis in the hypertrophic left ventricle of renin-dependent hypertensive rats: identification of target genes for renin.

INTRODUCTION: The aim of this work was to identify new renin target genes in left ventricular hypertrophy during hypertension. MATERIALS AND METHODS: We compared left ventricle gene expression from four transgenic TGR(mRen2)27 (TG+/-) rats and four non-transgenic littermates (TG-/-) using cDNA macroarray. Hybridisation signals were quantified with a phosphorimager, and normalised to an external scale. Data analysis was performed with Statistical Analysis for Microarrays (SAM 1.21) software. The mRNA levels of candidate genes were determined by semi-quantitative RT-PCR in three different hypertensive rats: TG+/-, spontaneously hypertensive (SHR) and genetically Lyon hypertensive (LH) rats, compared to their respective controls (TG-/-, Wistar-Kyoto, Lyon low blood pressure rats). RESULTS: Out of 1,200 genes present on the macroarray, 233 were reliably measured and only three were overexpressed (Biglycan, beta1-adenosine monophosphate-activated protein kinase [AMPK] and amyloid precursor like protein 2 [APLP2]) and 19 were underexpressed in the left ventricle of TG+/- compared with TG-/-. APLP2 is a member of the amyloid precursor protein (APP) family. APLP2 and APP mRNA levels were increased in TGR(mRen2)27 but significantly decreased in LH rats, while only APP was increased in SHR rats. CONCLUSIONS: We report new genes associated with renin-dependent left ventricular hypertrophy. Moreover, this work shows for the first time that the APP family gene expression could be altered in response to high renin activity and this effect is independent of cardiac remodelling and hypertension.

AMP-Activated Protein Kinases↗
PubMed2000

Photoactivatable retroviral vectors: a strategy for targeted gene delivery.

We have explored a novel strategy for the targeting of retroviral vectors to particular sites or cell types. This strategy involves a method whereby the infectivity of a retroviral vector is neutralized by treatment of viral particles with a photocleavable, biotinylation reagent. These modified viral vectors possess little to no infectivity for target cells. Exposure of these modified viral vectors to long-wavelength UV light induces a reversal of the neutralizing, chemical modification resulting in restoration of infectivity to the viral vector. This infectivity 'trigger' possesses great potential, both as a research tool and as a novel tactic for the targeting of gene-transfer agents, since it would become possible to direct both the time and location of a viral infection in a versatile manner.

Animals↗
PubMed2006

Down-regulation of PARP-1, but not of Ku80 or DNA-PKcs', results in higher gene targeting efficiency.

The viability of non-homologous end-joining (NHEJ)-defective mice suggests that homologous recombination (HR) might take over its role in DNA repair. To test this hypothesis, we examined gene targeting frequencies (TF) in DNA-PK(cs), Ku80 and poly(ADP-ribose) polymerase (PARP-1) nullizygous cells. We observed a 3-fold TF increase in PARP-1 knockout embryonic stem (ES) cells, which is consistent with the predicted role of PARP-1 as a switch between HR and NHEJ. To a lesser extent, such effect could be reproduced upon chemical inhibition of PARP-1. However, TF was not enhanced in Ku80- or DNA-PK(cs)-defective cells. Our study also suggests an unexpected involvement of DNA-PK(cs) in HR.

Animals↗
PubMed1991

Targeted gene walking polymerase chain reaction.

We describe a modification of a polymerase chain reaction method called 'targeted gene walking' that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific 'walking' primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.

Autoradiography↗
PubMed2005

FGF8, Wnt8 and Myf5 are target genes of Tbx6 during anteroposterior specification in Xenopus embryo.

The T-box transcription factor Tbx6 is required for somite formation and loss-of-function or reduced activity of Tbx6 result in absence of posterior paraxial mesoderm or disorganized somites, but how it is involved in a regulatory hierarchy during Xenopus early development is not clear. We show here that Tbx6 is expressed in the lateral and ventral mesoderm of early gastrula, and it is necessary and sufficient to directly and indirectly regulate the expression of a subset of early mesodermal and endodermal genes. Ectopic expression of Tbx6 inhibits early neuroectodermal gene expression by strongly inducing the expression of posterior mesodermal genes, and expands the mesoderm territory at the expense of neuroectoderm. Conversely, overexpression of a dominant negative Tbx6 mutant in the ventral mesoderm inhibits the expression of several mesodermal genes and results in neural induction in a dose-dependent manner. Using a hormone-inducible form of Tbx6, we have identified FGF8, Xwnt8 and XMyf5 as immediate early responsive genes of Tbx6, and the induction of these genes by Tbx6 is independent of Xbra and VegT. These target genes act downstream and mediate the function of Tbx6 in anteroposterior specification. Our results therefore identify a regulatory cascade governed by Tbx6 in the specification of posterior mesoderm during Xenopus early development.

Animals↗
PubMed2005

Identification of target genes in breast cancer cells directly regulated by the SRC-3/AIB1 coactivator.

Steroid receptor coactivator-3 (SRC-3/AIB1) is a coactivator for nuclear receptors and other transcription factors and an oncogene that contributes to growth regulation and development of mammary and other tumor types. Because of its biological functions, it is important to identify genes regulated by SRC-3. However, because coactivators do not bind DNA directly, extensive work is required to determine whether genes identified by RNA profiling approaches are direct or indirect targets. Here, we report the use of chromatin immunoprecipitation (ChIP)-based assays that involve genomic mapping and computational analyses of immunoprecipitated DNA to identify SRC-3-binding target genes in estradiol (E2)-treated MCF-7 breast cancer cells. We identified 18 SRC-3 genomic binding sites and demonstrated estrogen receptor-alpha (ERalpha) binding to all of them. Both E2-dependent and -independent SRC-3/ERalpha-binding sites were identified. RNA polymerase II ChIP assays were used to determine the correlation between SRC-3 and ERalpha binding and recruitment of the transcriptional machinery. These assays, in conjunction with analyses of RNA obtained from E2-treated cells, lead to the identification of SRC-3/ERalpha-associated genes. The ability of SRC family coactivators to regulate the expression of one of these genes, PARD6B/Par6, was confirmed by using cells individually depleted of SRC-1, SRC-2, or SRC-3 by small interfering RNA. The method described herein can be used to identify genes regulated by non-DNA-binding factors, such as other coactivators or corepressors, as well as DNA-binding transcription factors, and provides information on their binding location that can accelerate further gene characterization.

Acetyltransferases↗
PubMed1997

Epidermal muscle attachment site-specific target gene expression and interference with myotube guidance in response to ectopic stripe expression in the developing Drosophila epidermis.

The egr-type zinc-finger transcription factor encoded by the Drosophila gene stripe (sr) is expressed in a subset of epidermal cells to which muscles attach during late stages of embryogenesis. We report loss-of-function and gain-of-function experiments indicating that sr activity provides ectodermal cells with properties required for the establishment of a normal muscle pattern during embryogenesis and for the differentiation of tendon-like epidermal muscle attachment sites (EMA). Our results show that sr encodes a transcriptional activator which acts as an autoregulated developmental switch gene. sr activity controls the expression of EMA-specific target genes in cells of ectodermal but not of mesodermal origin. sr-expressing ectodermal cells generate long-range signals that interfere with the spatial orientation of the elongating myotubes.

Animals↗
PubMed2002

[Molecular conformation and expression efficiency in the hepatocyte targeting gene drug].

OBJECTIVE: To modulate the molecular conformation of the hepatocyte targeting drug to increase exogenous gene expression efficiency in the targeted cell. METHODS: Some specific auxiliary molecules were added to the gene drug its molecular liquid conformation was investigated under the electron-microscope and they were transfected into human hepatoma cell line BEL-7402 in vitro. We detected IFN-gamma gene expression product by ELISA and screened out the most efficient drug molecules. RESULTS: Due to various concentrations of auxiliary molecules, the conformation of hepatocyte targeting drug changed, including floccule, globulous, stringbeads, bacillform and chromosome-like mixture. While the most efficient expression conformation was the bacilliform and chromosome-like mixture and didn't need to inhibit the lysozyme activity, the expression efficiency of such drug molecules was much higher than that of the liposome targeting vector. CONCLUSION: The molecular drug conformation had much influence on targeted gene expression efficiency in gene therapy. Bacilliform and chromosome-like mixture may be the most efficient expression conformation to construct drug molecules in ASOR targeted hepatocyte gene therapy. So we put forward a new concept "chromosome mimic conformation" in screening out the most efficient molecular drug conformation in gene therapy.

Carcinoma, Hepatocellular↗
PubMed2006

[New option for gene-targeting vector construction Red/ET recombination].

The study of human new gene's function has become an increasingly active academic field basically relying on the gene knockout (KO) mouse. The construction of targeting vector economically and efficiently has turned into the key step to acquire a KO mouse because of the low efficiency of recombination with traditional constructed targeting vector. For study of the function of new gene-Resp18, we brought in a new DNA engineering platform-Red/ET recombination to construct Resp18 targeting vector. Red/ET recombineering differs from the conventional ways of vector construction (e.g., PCR, restriction enzyme digestion and ligation) and achieves genetic modification by acquisition, insertion, fusion or replacement of the target gene through small fragments mediated homologous recombination. Now Resp18 targeting vectors of three strategies were yielded successfully through two homologous recombination processes of retrieve and neo-targeting. Red/ET recombination has the advantage of getting longer homology regions without mutation, which makes it a new and reliable alternative to the construction of a targeting vector today.

Bacteriophage lambda↗
PubMed2004

In vivo targeted gene transfer in skin by the use of laser-induced stress waves.

BACKGROUND AND OBJECTIVES: Much interest has been shown in the use of lasers for nonviral targeted gene transfer, since the spatial characteristics of laser light are quite well defined. The aim of this study was to demonstrate in vivo gene transfer by the use of laser-induced stress waves (LISWs). STUDY DESIGN/MATERIALS AND METHODS: After reporter genes had been intradermally injected to rat skin in vivo, a laser target was placed on the gene-injected skin. LISWs were generated by the irradiation of an elastic laser target with 532-nm nanosecond laser pulses of a Q-switched Nd:YAG laser. RESULTS: Levels of luciferase activities for the skin exposed to LISWs were two orders of magnitude higher than those for the skin injected with naked DNA. Expressions of enhanced green fluorescent protein (EGFP) and beta-galactosidase were observed only in the area that was exposed to LISWs, and in addition, epidermal cells were selectively transfected. No major side effects were observed, and luciferase activity levels as high as 10(5) RLU per mg of protein were sustained even 5 days after gene transfer. CONCLUSION: Highly efficient and site-specific gene transfer can be achieved by applying a few pulses of nanosecond pulsed LISWs to rat skin in vivo.

Animals↗