GeneChip Results Research Articles

光照射の生物学的効果の機能ゲノム科学的解明

In order to elucidate the mechanism of anti-inflammatory effect of light irradiation, the human rheumatoid synoviocytes, MH7A, were treated with the proinflammatory cytokine interleukin (IL)-1β, and irradiated with linear polarized infrared light by Super Lizer ® (SL) or treated with the glucocorticoid, dexamethasone (DEX), then gene expressions were monitored using Affymetrix Gene Chip. Furthermore, Gene Chip results were analyzed using the signal transduction pathway coreddatabase (Ingenuity Pathway Analysis, IPA). SL and DEX irradiation suppressed IL-1β-induced IL-8 gene expression leveland inflammatory factors. Interestingly, DEX randomly altered many gene expressions, and not only reduced anti-inflammatoryfactors but also increased inflammatory factors, whereas SL only reduced gene expression of anti-inflammatory factors. Thesefindings may support the reason why light therapy has no side effects on the course of therapy.

Gastric Inflammation, Metaplasia, and Tumor Development in Gastrin–Deficient Mice

Gastrin deficiency and proton pump inhibitor treatment cause achlorhydria, which predisposes to disease. To elucidate the underlying molecular biology, we examined the changes in gastric gene expression in both types of achlorhydria. We also explored the associated changes in the gastric microflora and the long-term consequences of gastrin-deficient achlorhydria. Expression profiles were generated from gastric RNA from wild-type mice, gastrin knockout (KO) mice, gastrin KO mice after 1 week of gastrin infusion, and wild-type mice treated for 1 month with a proton pump inhibitor. The results were confirmed using real-time polymerase chain reaction and immunohistochemistry. Selective media were used to characterize the gastric microflora. The number of gastric bacteria was increased in both gastrin KO and PPI-treated mice. The expression profiles revealed activation of immune defense genes, interferon-regulated response genes, and intestinal metaplasia of the gastric mucosa. In young gastrin-deficient mice, gastrin infusions reversed the changes. Over time, the changes accumulated, became irreversible, and progressed into metaplasia and polyp development. Finally, the study showed that gastrin regulated the expression of genes encoding extracellular matrix proteins. Independently of gastrin, achlorhydria is associated with gastric bacterial overgrowth and intestinal gene expression patterns and is associated with predisposition to disease. Gastrin is therefore essential for prevention of gastric disease, mainly through control of acid secretion but to a lesser extent also through control of gastric gene expression. The gastrin-deficient mouse serves as a useful new model for gastric metaplasia and neoplasia.

Alterations in mRNA and Protein Following Spinal Cord Injury in Humans

PURPOSE: There is a rapid loss of muscle mass following spinal cord injury (SCI). There are no known treatment options available to attenuate losses in muscle mass, given the lack of understanding of how SCI affects signaling pathways within the musculoskeletal system in humans. This work expanded on a preliminary microarray study that explored global changes in gene expression to identify pathways affected by SCI. The purpose of this work was two fold: 1) To examine alterations in mRNA for components of the ubiquitin proteasome pathway (UPP), metallothioneins (MTs) and an inflammatory response gene (secretory leukocyte protease inhibitor: SLPI) 2d and 5d post-SCI and 2) To examine alterations in protein levels for components of the AKT and UPP pathway, MTs, and SLPI. METHODS: Biopsies were taken from the vastus lateralis muscle of 5 patients 2d and 5d post-SCI. Global changes in gene expression were analyzed using Affymetrix gene chips. mRNA levels of candidate genes selected from this analysis were confirmed via quantitative Real Time Polymerase Chain Reaction (qRT-PCR). When alterations in mRNA were confirmed via qRT-PCR, Western blotting (WB) was used to assess protein products of candidate genes and components of the AKT pathway. Immunohistochemistry (IHC) was then used to confirm alterations in protein products observed via WB. RESULTS: 50 genes met inclusion criteria for gene chip analysis (fold-change >2.0; p <0.001; chronically up or down regulated at 2d and 5d). UPP components (UBE2E, PSMD11, Atrogin-1) were upregulated 2.0 to 11.3-fold. MTs (1F, 1H, 1A) were upregulated 3.9 to 20.7-fold, and SLPI was upregulated 44-fold. qRT-PCR confirmed gene chip results. qRT-PCR was used to explore the expression of MuRF-1, a component of the UPP shown to be dramatically upregulated in animals post-SCI. MuRF-1 expression was increased 4.9–6.5-fold. WB revealed that total AKT was unchanged but AKTser473 was decreased at 2d post-SCI (34%, p<0.05) but not 5d. MT and PSMD11 protein increased, 16 and 18% respectively, by 5d post-SCI. IHC analysis confirmed WB results. CONCLUSIONS: This work identifies specific signaling pathways that are affected in the first days post-SCI, likely contributing to losses in muscle mass in humans. The observed temporal pattern of change in mRNA and protein suggest that following SCI, there is an initial stress (decreased AKTser473, increased MT) and inflammatory response (SLPI), which is followed by increased activity of the UPP. These alterations are important biomarkers for future work investigating ways to manipulate these pathways post-SCI to attenuate losses in muscle mass. Acknowledgements: Christopher Reeve Paralysis Foundation.

Serial Gene Expression Profiling in the Intact Human Heart

In chronic heart failure due to a dilated cardiomyopathy phenotype, the molecular bases for contractile dysfunction and chamber remodeling remain largely unidentified. To investigate the feasibility of measuring global gene expression serially in the intact failing human heart, we performed repeated messenger RNA (mRNA) expression profiling using RNA extracted from endomyocardial biopsy specimens and gene chip methodology in 8 subjects with idiopathic dilated cardiomyopathy. In patients treated with beta-blocking agents or placebo, myocardial gene expression was measured in endomyocardial biopsy material and radionuclide ejection fraction was measured at baseline and after 4 to 12 months of treatment. Gene expression was measured for 12,625 gene sequences by using Affymetrix U95 gene chips and commercially available software. For 6 mRNAs, gene chip results were compared with measurements made by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In an unfiltered composite analysis of changes in expression detected in the patients with high-signal intensity chips, 241 genes showed an increase and 331 genes a decrease in mRNA abundance. There was good agreement between changes measured by quantitative RT-PCR and those determined by gene chips. There was less variance between differences in phenotype in patients sampled serially as compared between subjects with similar phenotypes sampled at baseline. Serial gene expression profiling with association to phenotypic change is feasible in the intact human heart and may offer advantages to cross-sectional expression profiling. This study suggests that the intact failing remodeled human heart is in an activated state of gene expression, with a large net reduction in gene expression occurring as phenotypic improvement occurs.

Human papillomavirus genotyping by a polymerase chain reaction-based genechip method in cervical carcinoma treated with neoadjuvant chemotherapy plus radical surgery.

The aim of this study was to evaluate the accuracy of human papillomavirus (HPV) genotyping by a polymerase chain reaction (PCR)-based genechip method and to determine the prognostic value of HPV genotype in bulky stage IB or IIA cervical carcinoma treated with neoadjuvant chemotherapy (NAC) and radical surgery. A total of 149 patients had adequate tissue for the study. The SPF1/GP6+ primers were used to amplify a 184 bp fragment. The amplimers were submitted for direct sequencing and hybridization with a genechip using revert-blot detection of 39 types of HPV DNA in a single reaction. Two runs of PCR with respective hybridization were performed for each tumor. The complete concordance of HPV genotyping was 80.5% (120/149) of the paired genechip results. The kappa coefficient was 0.634 (P < 0.0001). HPV DNA sequences were detected in 100% of the specimens, among which 67.8% harbored single type and 32.2% contained multiple types. HPV-16 was detected in 98.7%, HPV-18 in 22.8%, HPV-31 in 0.7%, HPV-45 in 1.3%, HPV-52 in 2.0%, HPV-58 in 6.7%, HPV-59 in 4.7%, and HPV-67 in 0.7%. In multivariate analyses, the HPV genotype [HPV-18 or HPV-16 and HPV-18 only versus all others: relative risk (RR), 2.33; 95% CI, 1.17-4.64; P = 0.016] and pre-NAC tumor size (>5 versus </=5 cm: RR, 2.25; 95% CI, 1.13-4.48; P = 0.021) were significantly related to overall survival. This PCR-based genechip method is sensitive and reproducible for HPV genotyping. The association of HPV-18 or HPV-16 and HPV-18 with poor outcome in cervical carcinoma treated with NAC plus radical surgery is confirmed.

Construction of a genechip method for detection of hepatitis B virus DNA, basal core promotor and Pre-C mutants

To establish a genechip method for detection of hepatitis B virus (HBV) DNA, basal core promotor (BCP), and Pre-C mutants. This study used two kinds of technology (PCR, oligochip), which can detect five mutant hotspots including nt 1 896, nt 1 899, nt 1 862, nt 1 764 and nt 1 762. The results of genechip method was verified by DNA sequencing. In detecting HBV DNA, the results of genechip were 100% consistent with those of DNA sequencing. In detecting HBV BCP and Pre-C mutants, 146 codons showed the same results using both methods, except for only 4 codons (P greater than 0.05). This convenient high throughput genechip method could detect several BCP and Pre-C mutant codons at the same time. These results suggest that genechip method has the same positive rate and specificity with DNA sequencing method. It has more advantages than the latter in detecting mixed mutants and therefore may be used in clinical practice.

Human papillomavirus genotyping by a polymerase chain reaction-based genechip method in cervical carcinoma treated with neoadjuvant chemotherapy plus radical surgery

The aim of this study was to evaluate the accuracy of human papillomavirus (HPV) genotyping by a polymerase chain reaction (PCR)-based genechip method and to determine the prognostic value of HPV genotype in bulky stage IB or IIA cervical carcinoma treated with neoadjuvant chemotherapy (NAC) and radical surgery. A total of 149 patients had adequate tissue for the study. The SPF1/GP6+ primers were used to amplify a 184 bp fragment. The amplimers were submitted for direct sequencing and hybridization with a genechip using revert-blot detection of 39 types of HPV DNA in a single reaction. Two runs of PCR with respective hybridization were performed for each tumor. The complete concordance of HPV genotyping was 80.5% (120/149) of the paired genechip results. The kappa coefficient was 0.634 (P&lt; 0.0001). HPV DNA sequences were detected in 100% of the specimens, among which 67.8% harbored single type and 32.2% contained multiple types. HPV-16 was detected in 98.7%, HPV-18 in 22.8%, HPV-31 in 0.7%, HPV-45 in 1.3%, HPV-52 in 2.0%, HPV-58 in 6.7%, HPV-59 in 4.7%, and HPV-67 in 0.7%. In multivariate analyses, the HPV genotype [HPV-18 or HPV-16 and HPV-18 only versus all others: relative risk (RR), 2.33; 95% CI, 1.17–4.64;P= 0.016] and pre-NAC tumor size (&gt;5 versus ≤5 cm: RR, 2.25; 95% CI, 1.13–4.48;P= 0.021) were significantly related to overall survival. This PCR-based genechip method is sensitive and reproducible for HPV genotyping. The association of HPV-18 or HPV-16 and HPV-18 with poor outcome in cervical carcinoma treated with NAC plus radical surgery is confirmed.

Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant.

Oligomer-based DNA Affymetrix GeneChips representing about one-third of Arabidopsis (Arabidopsis thaliana) genes were used to profile global gene expression in a single cell type, guard cells, identifying 1309 guard cell-expressed genes. Highly pure preparations of guard cells and mesophyll cells were isolated in the presence of transcription inhibitors that prevented induction of stress-inducible genes during cell isolation procedures. Guard cell expression profiles were compared with those of mesophyll cells, resulting in identification of 64 transcripts expressed preferentially in guard cells. Many large gene families and gene duplications are known to exist in the Arabidopsis genome, giving rise to redundancies that greatly hamper conventional genetic and functional genomic analyses. The presented genomic scale analysis identifies redundant expression of specific isoforms belonging to large gene families at the single cell level, which provides a powerful tool for functional genomic characterization of the many signaling pathways that function in guard cells. Reverse transcription-PCR of 29 genes confirmed the reliability of GeneChip results. Statistical analyses of promoter regions of abscisic acid (ABA)-regulated genes reveal an overrepresented ABA responsive motif, which is the known ABA response element. Interestingly, expression profiling reveals ABA modulation of many known guard cell ABA signaling components at the transcript level. We further identified a highly ABA-induced protein phosphatase 2C transcript, AtP2C-HA, in guard cells. A T-DNA disruption mutation in AtP2C-HA confers ABA-hypersensitive regulation of stomatal closing and seed germination. The presented data provide a basis for cell type-specific genomic scale analyses of gene function.

Distinct patterns of chemokine gene expression in the skin lesions of atopic dermatitis and psoriasis: A gene microarray analysis

Rationale Atopic dermatitis (AD) and psoriasis are the two most common chronic inflammatory skin diseases. Both of these diseases have distinct clinical findings and specific inflammatory cell infiltrates. Previous reports have focused individually on one or two chemokine genes or gene products in the lesions of both skin diseases. However, they have not captured the complex gene expression that must occur to induce specific cellular infiltrates in the skin lesions of these two diseases. DNA microarray studies allow the simultaneous comparison of thousands of messenger RNAs which may identify the disease-specific pattern of tissue inflammatory responses. Methods RNA was extracted from skin biopsies of six AD and seven psoriasis patients, and analyzed using Hu-U95Av. GeneChip microarrays. To confirm GeneChip results, real-time PCR of selected genes were performed. Results In AD skin, a total of 18 genes including the CC-chemokines, CCL-13/MCP-4, CCL-18/PARC and CCL-27/CTACK showed a statistically significant, greater than two-fold increase of gene expression compared to psoriasis. In psoriasis skin, a total of 59 genes including CCL-4/MIP-1β, CCL-20/MIP-3α, CXCL-2/GRO-β, CXCL-8/IL-8 and CXCR2/IL-8R showed a greater than two-fold increase of gene expression compared to AD skin. Real-time PCR confirmed several of these GeneChip results. Conclusions These results show a very distinctive gene expression pattern in AD as compared to psoriasis that may explain the specific inflammatory cell infiltrates observed in these disorders, i.e. Th2 cells, eosinophils and mast cells in AD; Th1 cells and neutrophils in psoriasis.

Distinct patterns of gene expression in the skin lesions of atopic dermatitis and psoriasis: A gene microarray analysis

Background Atopic dermatitis (AD) and psoriasis are the two most common chronic inflammatory skin diseases. Both of these diseases have distinct clinical findings and specific inflammatory cell infiltrates. Previous reports have focused individually on one or two genes or gene products in the lesions of both skin diseases. However, they have not captured the complex gene expression that must occur to induce specific cellular infiltrates in the skin lesions of these two diseases. DNA microarray studies allow the simultaneous comparison of thousands of messenger RNAs that may identify the disease-specific pattern of tissue inflammatory responses. Objective To compare the complex gene expression pattern of AD versus psoriasis skin lesions. Methods RNA was extracted from skin biopsy specimens of 6 patients with AD and 7 patients with psoriasis and analyzed with the use of Hu-U95Av.GeneChip microarrays. To confirm GeneChip results, real-time PCR of selected genes were performed. Results In AD skin, a total of 18 genes including the CC chemokines, CCL-13/MCP-4, CCL-18/PARC, and CCL-27/CTACK showed a statistically significant, >2-fold increase of gene expression compared with psoriasis. In psoriasis skin, a total of 62 genes including CCL-4/MIP-1β, CCL-20/MIP-3α, CXCL-2/GRO-β CXCL-8/IL-8, and CXCR2/IL-8R showed a >2-fold increase of gene expression compared with AD skin. Real-time PCR confirmed several of these GeneChip results. Conclusions These results show a very distinctive gene expression pattern in AD as compared with psoriasis that may explain several features of AD and psoriasis including the specific inflammatory cell infiltrates observed in these disorders, that is, T H2 cells, eosinophils, and mast cells in AD and T H1 cells and neutrophils in psoriasis. Such observations may contribute to a characteristic “signature” for these two skin diseases.

Gene expression profile in diabetic KK/Ta mice

To identify susceptibility genes for diabetic nephropathy, GeneChip Expression Analysis was employed to survey the gene expression profile of diabetic KK/Ta mouse kidneys. Kidneys from three KK/Ta and two BALB/c mice at 20 weeks of age were dissected. Total RNA was extracted and labeled for hybridizing to the Affymetrix Murine Genome U74Av2 array. The gene expression profile was compared between KK/Ta and BALB/c mice using GeneChip expression analysis software. Competitive reverse transcription-polymerase chain reaction (RT-PCR) was used to confirm the results of GeneChip for a selected number of genes. Out of 12,490 probe pairs present on GeneChip, 98 known genes and 31 expressed sequence tags (ESTs) were found to be differentially expressed between KK/Ta and BALB/c kidneys. Twenty-one known genes and seven ESTs that increased in expression and 77 known genes and 24 ESTs that decreased in KK/Ta kidneys were identified. These genes are related to renal function, extracellular matrix expansion and degradation, signal transduction, transcription regulation, ion transport, glucose and lipid metabolism, and protein synthesis and degradation. In the vicinity of UA-1 (quantitative trait locus for the development of albuminuria in KK/Ta mice), candidate genes that showed differential expression were identified, including the Sdc4 gene for syndecan-4, Ahcy gene for S-adenosylhomocysteine hydrolase, Sstr4 gene for somatostatin receptor 4, and MafB gene for Kreisler leucine zipper protein. The gene expression profile in KK/Ta kidneys is different from that in age-matched BALB/c kidneys. Altered gene expressions in the vicinity of UA-1 may be responsible for the development of albuminuria in diabetic KK/Ta mice.

Gene expression profile after intense second messenger activation in cortical primary neurones.

Numerous stimuli induce immediate early gene (IEG) expression in neurones, but a comprehensive overview of the late-response genes is lacking. Therefore we aimed to identify changes in the neuronal gene expression profile following intense stimulation. Forskolin and 12-O-tetradecanoylphorbol-13-acetate (TPA), direct activators of intracellular second messengers, were applied to primary cultured cortical neurones. The gene expression profiles were analyzed on Affymetrix DNA chips which cover around 8000 rat genes. Out of these, 95 genes (1.2%) were increased at least three-fold, and 43 genes (0.5%) were at least three-fold decreased. The gene chip results were verified by testing 15 of the altered genes by quantitative real-time PCR. The majority of the up-regulated genes were transcription factors, neurotrophic factors or (putative) neuropeptides. Furthermore, there were marked changes in intracellular signal processing enzymes and in postsynaptic structural proteins (e.g. vesl, arc, narp), which have been implicated in synaptic plasticity. Notably, classical players in neurotransmission or plasticity such as glutamate and GABA receptors or voltage-gated ion channels were not increased. It is likely that the increased production of components of intracellular signalling and of postsynaptic proteins is involved in neuronal plasticity.