Significant Decrease In Gene Expression Research Articles

Neuroblastoma tumors with favorable and unfavorable outcomes: Significant differences in mRNA expression of genes mapped at 1p36.2

The distal part of 1p is frequently deleted in aggressive neuroblastoma, and the region is believed to harbor one or more tumor suppressor genes relevant to tumor development. To analyze differences among neuroblastoma tumors, an expression profile was established for the genes mapped within a previously described shortest region of overlap of deletions at 1p36.2. The gene expression levels were quantified by TaqMan real-time (RT)-PCR for 30 transcripts using 55 primary neuroblastoma tumors. Here we report on a significant decrease in gene expression of the genes RERE, PIK3CD, LZIC, PGD, and PEX14 and an increase of SLC2A5 when comparing tumors of favorable biology to Stage 4 neuroblastomas. When comparing 1p-deleted tumors of all stages to tumors with an intact 1p, a significant difference at gene-by-gene level in TNFRSF9, RERE, PIK3CD, CLSTN1, CTNNBIP1, and CASZ1 was detected. A complete loss of expression could not be seen for any single gene analyzed. Several of the genes with diminished expression in unfavorable or 1p-deleted tumors have functions that could contribute to tumor development. It is also possible that a combination of lowly expressed genes at 1p, rather than one single classical tumor suppressor gene, causes the unfavorable outcome associated with 1p-deletion in neuroblastoma.

Transcriptional analysis of protective antigen-stimulated PBMC from non-human primates vaccinated with the anthrax vaccine absorbed

The transcriptional responses in recombinant protective antigen (PA)-stimulated peripheral blood mononuclear cells (PBMCs) from Anthrax Vaccine Absorbed (AVA)-vaccinated rhesus macaques were evaluated using Affymetrix HGU133 Plus 2.0 GeneChips. PBMCs from animals vaccinated at 0, 4, and 26 weeks were harvested at week 30, stimulated with PA, and RNA isolated. The expression of 295 unigenes was significantly increased in PA-stimulated compared to non-stimulated PBMCs; no significant decrease in gene expression was observed. These upregulated transcripts encoded for proteins functioning in both innate and adaptive immunity. Results were corroborated for several genes by real-time RT-PCR. This study provides information on the potential underlying transcriptional mechanisms in the immune response to PA in AVA-vaccinated rhesus macaques.

592. Transfection of Human Oral Cancer Cells Mediated by Transferrin-Associated Lipoplexes: Mechanisms of Cell Death Induced by Herpes Simplex Virus Thimidine Kinase/Ganciclovir Therapy

The Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene/ganciclovir approach has been used for the treatment of a variety of cancers. Cells that express HSV-tk are rendered sensitive to the pro-drug ganciclovir which is phosphorylated into a toxic agent. Furthermore, there may be another advantage of HSV-tk/GCV therapy. Both HSV-tk-expressing and non-expressing cells can be killed by the bystander effect which allows the possibility of complete tumor eradication even at relatively low levels of cell transfection. Different mechanisms have been proposed to explain this effect: the formation of apoptotic vesicles containing the toxic metabolite, which are internalized by neighboring cells, the activation of immune system in vivo, and diffusion of the toxic metabolite into non-transfected cells trough gap junctions.The purpose of this study was to evaluate the cytotoxic effect of ganciclovir in human tongue cancer cells (HSC-3) previously transfected with transferrin-associated lipoplexes containing DOTAP/cholesterol as well as to investigate the mechanisms involved in the process of cell death and the bystander effect. To optimize the transfection conditions, a plasmid coding for the reporter gene luciferase was used, and the levels of gene expression assessed by luminescence measurements. Our results show that the highest efficiency of transfection was obtained upon association of transferrin (Tf) to lipoplexes prepared at 3/2 (+/-) charge ratio. The delivery of HSV-tk gene to HSC-3 cells mediated by this Tf-lipoplexes followed by ganciclovir treatment for 7 days resulted in 90% cytotoxicity, as assessed by Alamar Blue assay. Cell death was analysed by flow cytometry, by staining the cells with FITC-Annexin V, and propidium iodide. We observed that cell death occur mainly by an apoptotic process. Since only a low number of cells were efficiently transfected, the high cytotoxicity obtained can be attributed to a bystander effect. The absence of any toxic metabolite in the cell culture medium collected from transfected cells followed by incubation with ganciclovir, and the significant decrease in gene expression observed 2 days after transfection, suggest that the observed cytotoxicity was due to diffusion of the toxic agent into neighbouring cells via gap junctions. In order to study the role of gap junctions in the bystander effect, we used dibutyryl-cAMP as a gap junction inducer and α-glycyrrhetinic acid as a gap junction blocker. At five days incubation, cell survival was only reduced to 60 % (50 μM AGA) as compared to 25% in the absence of AGA when transfected cells were treated with 50 μM GCV. On other hand db-cAMP promoted the bystander effect. Cytotoxicity of 25 μM ganciclovir was increased from 30% to 70% when cells were pre-treated with 500 mM db-cAMP. Our results indicate that the observed toxic effect is GCV dose-dependent. Diffusion of the toxic agent into neighbouring cells via gap junctions is most likely involved in the process of cell death which occurs mainly by an apoptotic process. Overall, we can conclude that Tf-associated lipoplexes constitute a promising tool for suicide gene therapy of oral carcinomas

Analysis of a human brain transcriptome map.

BackgroundGenome wide transcriptome maps can provide tools to identify candidate genes that are over-expressed or silenced in certain disease tissue and increase our understanding of the structure and organization of the genome. Expressed Sequence Tags (ESTs) from the public dbEST and proprietary Incyte LifeSeq databases were used to derive a transcript map in conjunction with the working draft assembly of the human genome sequence.ResultsExamination of ESTs derived from brain tissues (excluding brain tumor tissues) suggests that these genes are distributed on chromosomes in a non-random fashion. Some regions on the genome are dense with brain-enriched genes while some regions lack brain-enriched genes, suggesting a significant correlation between distribution of genes along the chromosome and tissue type. ESTs from brain tumor tissues have also been mapped to the human genome working draft. We reveal that some regions enriched in brain genes show a significant decrease in gene expression in brain tumors, and, conversely that some regions lacking in brain genes show an increased level of gene expression in brain tumors.ConclusionsThis report demonstrates a novel approach for tissue specific transcriptome mapping using EST-based quantitative assessment.

Aroclor 1254 alters morphology, survival, and gene expression in Xenopus laevis tadpoles.

PCBs are persistent environmental contaminants that cause a variety of adverse health effects in wildlife and humans. This article describes the use of signature gene expression patterns that link increased PCB exposure with progressive, adverse biological effects. Developing Xenopus laevis tadpoles of two age classes were exposed to the PCB mixture Aroclor 1254 for 2 days. Real-time PCR was used to quantitate mRNA expression for 11 physiologically relevant, potential bioindicator genes. Younger tadpoles (5 days postfertilization) were resistant to Aroclor 1254 and showed few changes in gross morphology, swimming behavior, survival, or gene expression. Older tadpoles (11 days postfertilization) were more susceptible to Aroclor 1254. Exposure to 25 and 50 ppm Aroclor 1254 caused alterations in gross morphology and swimming behavior and statistically significant decreases in survival. These tadpoles showed statistically significant decreases in gene expression for 9 out of the 11 genes measured. Tadpoles exposed to 10 ppm showed incipient health changes but had gene expression profiles similar to the tadpoles treated with higher doses of Aroclor 1254. Tadpoles exposed to 1 ppm did not exhibit any observable adverse health effects, yet statistically significant decreases in gene expression occurred in these tadpoles (4 out of 11 genes). After prolonged exposure, tadpoles exposed to 1 and 10 ppm Aroclor 1254 exhibited health effects similar to those exposed to higher concentrations. Therefore, changes in expression of specific genes may serve not only as molecular bioindicators of Aroclor 1254 exposure but also as predictors of impending adverse health effects.

Enhanced Cutaneous Gene Delivery Following Intradermal Injection of Naked DNA in a High Ionic Strength Solution

Intradermal injection of naked DNA results in gene transfer to skin cells, but the efficiency of this gene transfer method is relatively low and variable. We have systematically optimized several parameters to obtain reproducible, high-level gene transfer to the mouse skin. Older mice (∼7 weeks) showed a significant decrease in gene expression compared with younger mice (4–5 weeks old). The composition of the solvent vehicle (electrolyte versus nonelectrolyte) strongly affected gene expression in the skin. A higher level of gene expression was achieved when naked DNA was dissolved in isotonic phosphate buffered saline solution compared with isotonic dextrose solution. Finally, transfection efficiency in older mice was greatly improved by increasing the ionic strength of the solvent vehicle. The improved transfection efficiency was due to an enhanced DNA uptake by the skin cells. Gene transfer was most evident in the subdermal smooth muscle cells and epidermal cells. With the optimized conditions, gene transfer mediated by intradermal injection of naked DNA was comparable in efficiency to electroporation. However, cellular distributions of the gene transfer of the two methods were different.

Evaluation of the role of lipoprotein metabolism genes in systemic cationic liposome-mediated gene transfer in vivo.

Germ line gene disruption and gene insertion are often used to study the function of selected genes in vivo. We used selected knockout and transgenic mouse models to attempt to identify lipoprotein-related genes and gene products that regulate the process of intravenous cationic liposome-DNA complex (CLDC)-based gene delivery. Several observations suggested that proteins involved in lipoprotein metabolism might be important in influencing the delivery and/or expression of CLDC. First, in vitro transfection of either K562 or CHO cells by CLDCs was enhanced by the presence of a functional low-density lipoprotein receptor (LDLR). Second, pretreatment of mice with 4-aminopyrazolopyrimidine (4APP), an agent that alters lipoprotein profiles in mice, significantly decreased expression of luciferase (luc) after intravenous injection of CLDC-luc complexes in mice. Therefore, we tested mouse model systems either deficient for, or overexpressing, selected genes involved in lipoprotein metabolism, for their potential to regulate intravenous, CLDC-based gene delivery. Although homozygous knockout mutation in the apoE gene caused a significant decrease in gene expression in many tissues of apoE-deficient mice, mice with homozygous deletion of both the apoE and LDLR genes showed wild-type levels of gene transfer efficiency. Thus, a secondary event, produced by homozygous deletion of apoE, but compensated for by the concomitant deletion of LDLR, and/or effects resulting from strain-related, genetic background differences, appeared to play a significant role in mediating intravenous, CLDC-based gene delivery. Secondary alterations resulting from germ line knockouts, as well as epigenetic effects produced by strain differences, may limit the ability to assign specific, gene transfer-related functions to the deleted gene.

The introduction of an inverted repeat to the 5′ untranslated leader sequence of a transgene strongly inhibits gene expression

Stable secondary structures in naturally occurring 5' untranslated regions have been shown to down-regulate both transcription and translation. We introduced a synthetic GC-rich inverted repeat to the leader sequence of a transgene to determine its influence on gene expression. The addition of a 54-bp inverted repeat led to a more than 90% reduction in transient gene expression, while the addition of an inverted repeat of 42-bp reduced gene expression by 88%. Complete removal of the inverted repeat abolished this inhibiting effect. This dramatic decrease in transgene expression is probably due to the formation of stable stem-loop structures in the 5' untranslated region of the reporter gene. The secondary structure energy of the putative stem-loop structures in the 54-bp and 42-bp repeats are -64.6 and -40.3 kcal mol-1, respectively. In comparison, the most stable stem-loop structure in the control construct's leader has a free energy of -15.4 kcal mol-1. This has important implications for the design of expression vectors where the recombination of multiple cloning sites and other 5' leader sequences can lead to the introduction of inverted repeats that has the potential of forming stable stem-loop structures and resulting in a significant decrease in gene expression.

Treatment of inflammatory bowel disease in a rodent model with the intestinal growth factor glucagon-like peptide-2

Background/Purpose: Microinjection of a Fisher (F344) rat zygote with human HLA-B27 and β2-microglobulin genes induces spontaneous chronic gastrointestinal (GI) inflammation similar to lesions seen in patients with inflammatory bowel disease (IBD). This study was designed to evaluate the potential therapeutic benefit of GLP-2, an intestinal growth factor, in this transgenic rat model of IBD. Methods: Five F344 (control) and 10 HLA-B27 (on a F344 background) rats at 25 weeks of age were used. Rats were divided into the following 3 groups: group 1, F344 rats, no treatment (n = 5); group 2, HLA-B27, no treatment (n = 5); and group 3, HLA-B27, treated with a 14-day systemic infusion (via the jugular vein) of GLP-2 at 50 μg/kg/d (n = 5). After infusion, all rats underwent laparotomy, and the intestine from the ligament of Treitz to the rectum was harvested. Total mucosal damage (percent surface area) was measured using image analysis software (Sigmascan 2.0). Microscopic analysis was performed by a blinded reviewer and scored as follows: 0, no inflammation; 1, mild inflammation; 2, moderate inflammation; and 3, severe inflammation. Colonic mucosal total RNA was assayed for tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-2 (IL-2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), internal standard, mRNA by reverse transcriptase polymerase chain reaction. Statistical analysis was performed using analysis of variance (ANOVA) and expressed as mean ± SEM. Results: Normal F344 rats did not show evidence of gross or histological lesions in the small or large intestine. GLP-2 reduced total mucosal damage from 9.0% ± 0.7% in group 2 to 0.9% ± 0.5% in group 3 (P <.01). The histological lesion score was reduced from 7.0 ± 0.6 in group 2 to 4.4 ± 0.8 in group 3 (P <.01). Furthermore, GLP-2 reduced the mean band intensity (MBI) of TNF-α (0.4 ± 0.04 in group 2 to 0 in group 3, P <.01) and IFN-γ (0.3 ± 0.02 in group 2 to 0 in group 3, P <.01). Conclusions: These data show for the first time that GLP-2 significantly reduces gross (90% decrease) and histological (40% decrease) lesions in this rat model of IBD. This is further supported by a significant decrease in gene expression of the inflammatory mediators TNF-α (100% decrease) and IFN-γ (100% decrease). These data suggest a potential therapeutic role for GLP-2 in IBD. J Pediatr Surg 35:847-851. Copyright © 2000 by W.B. Saunders Company.